103P2D6: tissue specific protein highly expressed in various cancers

ABSTRACT

A novel gene (designated 103P2D6) and its encoded protein are described. 103P2D6 is not expressed in normal adult tissue, but is highly expressed in prostate tissue xenografts, providing evidence that it is turned on in prostate cancer. 103P2D6 is also expressed in some fetal tissues, and in breast, bladder, lung, bone, colon, pancreatic, testicular, cervical and ovarian cancers. Consequently, 103P2D6 provides a diagnostic and/or therapeutic target for cancers, and the 103P2D6 gene or fragment thereof, or its encoded protein or a fragment thereof can be used to elicit an immune response.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/793,451 filed 26Feb. 2001, now abandoned, which application claims the benefit of U.S.provisional application 60/184,558, filed 24 Feb. 2000 and U.S.provisional application 60/218,856, filed 13 Jul. 2000. The contents ofthese applications are incorporated herein by reference.

FIELD OF THE INVENTION

The invention described herein relates to a novel gene and its encodedprotein, termed 103P2D6, and to diagnostic and therapeutic methods andcompositions useful in the management of various cancers that express103P2D6.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, cancer causes the death of well over ahalf-million people annually, with some 1.4 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise. Inthe early part of the next century, cancer is predicted to become theleading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most commoncancer in males and is the second leading cause of cancer death in men.In the United States alone, well over 40,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, surgical castration and chemotherapy continue to be the maintreatment modalities. Unfortunately, these treatments are ineffectivefor many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the diagnosis and management of this disease. Although theserum prostate specific antigen (PSA) assay has been a very useful tool,however its specificity and general utility is widely regarded aslacking in several important respects.

Progress in identifying additional specific markers for prostate cancerhas been improved by the generation of prostate cancer xenografts thatcan recapitulate different stages of the disease in mice. The LAPC (LosAngeles Prostate Cancer) xenografts are prostate cancer xenografts thathave survived passage in severe combined immune deficient (SCID) miceand have exhibited the capacity to mimic the transition from androgendependence to androgen independence (Klein et al., 1997, Nat.Med.3:402). More recently identified prostate cancer markers includePCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252),prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res1996 Sep;2(9):1445–51), STEAP (Proc Natl Acad Sci U S A. Dec. 71999;96(25):14523–8) and prostate stem cell antigen (PSCA) (Reiter etal., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

While previously identified markers such as PSA, PSM, PCTA and PSCA havefacilitated efforts to diagnose and treat prostate cancer, there is needfor the identification of additional markers and therapeutic targets forprostate and related cancers in order to further improve diagnosis andtherapy.

SUMMARY OF THE INVENTION

The present invention relates to a novel gene, designated 103P2D6 thatis over-expressed in multiple cancers listed in Table I. Northern blotexpression analysis of 103P2D6 gene expression in normal tissues shows arestricted expression pattern in adult tissues. Analysis of 103P2D6expression in normal prostate and prostate tumor xenografts showsover-expression in LAPC-4 and LAPC-9 prostate tumor xenografts. Thenucleotide (FIG. 2) and amino acid (FIG. 2 and FIG. 3) sequences of103P2D6 are provided. Portions of the 103P2D6 amino acid sequence showsome homologies to ESTs in the dbEST database. The tissue-relatedprofile of 103P2D6 in normal adult tissues, combined with theover-expression observed in prostate and other tumors, shows that103P2D6 is aberrantly over-expressed in at least some cancers, and thusserves as a useful diagnostic and/or therapeutic target for cancers ofthe tissues listed in Table I.

The invention provides polynucleotides corresponding or complementary toall or part of the 103P2D6 genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding103P2D6-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25amino acids; as well as the peptides/proteins themselves; DNA, RNA,DNA/RNA hybrids, and related molecules, polynucleotides oroligonucleotides complementary or having at least a 90% homology to the103P2D6 genes or mRNA sequences or parts thereof, and polynucleotides oroligonucleotides that hybridize to the 103P2D6 genes, mRNAs, or to103P2D6-encoding polynucleotides. Also provided are means for isolatingcDNAs and the genes encoding 103P2D6. Recombinant DNA moleculescontaining 103P2D6 polynucleotides, cells transformed or transduced withsuch molecules, and host-vector systems for the expression of 103P2D6gene products are also provided. The invention further providesantibodies that bind to 103P2D6 proteins and polypeptide fragmentsthereof, including polyclonal and monoclonal antibodies, murine andother mammalian antibodies, chimeric antibodies, humanized and fullyhuman antibodies, and antibodies labeled with a detectable marker.

The invention further provides methods for detecting the presence andstatus of 103P2D6 polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express 103P2D6.A typical embodiment of this invention provides methods for monitoring103P2D6 gene products in a tissue or hematology sample having orsuspected of having some form of growth dysregulation such as cancer.

The invention further provides various immunogenic or therapeuticcompositions and strategies for treating cancers that express 103P2D6such as prostate cancers, including therapies aimed at inhibiting thetranscription, translation, processing or function of 103P2D6 as well ascancer vaccines.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. shows the 103P2D6 suppression subtractive hybridization (SSH)DNA sequence (SEQ ID NO: 3).

FIGS. 2A–B. shows the nucleotide and amino acid sequences of 103P2D6.cDNA and ORF for 103P2D6 clone B, Kozak sequence and start methionineare indicated in bold. See Example 2, infra.

FIG. 3. shows the amino acid sequence encoded by the open reading frameshown in FIG. 2, and lists the amino acid positions used forproteins/peptides of the invention. The 103P2D6 signal sequence isboxed.

FIG. 4. shows the sequence alignment of 103P2D6 (ORF from clone B) withenv protein from human endogenous retroviral HERV-H (FASTA accession:Q9UNM3). The 103P2D6 protein sequence has homology to the HERV-H envprotein (24.9% identity and 32.8% homology taking account of any gaps).

FIG. 5A–C. shows the northern blot analysis of 103P2D6 expression invarious normal human tissues (using the 103P2D6 SSH fragment as a probe)and LAPC xenografts. Two multiple tissue northern blots (Clontech) and axenograft northern blot were probed with the 103P2D6 SSH fragment. Sizestandards in kilobases (kb) are indicated on the side. Each lanecontains 2 μg of mRNA for the normal tissues and 10 μg of total RNA forthe xenograft tissues. The results show the expression of 103P2D6 inLAPC xenografts, and not in normal prostate and other tissues. Lanes inFIG. 5A represent (1) heart; (2) brain; (3) placenta; (4) lung; (5)liver; (6) skeletal muscle; (7) kidney; (8) pancreas. Lanes in FIG. 5Brepresent (1) spleen; (2) thymus; (3) prostate; (4) testis; (5) ovary;(6) small intestine; (7) colon; (8) leukocytes. Lanes in FIG. 5Crepresent (1) prostate; (2) LAPC-4 AD; (3) LAPC-4 AI; (4) LAPC-9 AD; (5)LAPC-9 AI.

FIG. 6 shows the northern blot analysis of 103P2D6 expression in variouscancer cell lines. Lanes represent (1) LAPC-4 AD; (2) LAPC-4 AI; (3)LAPC-9 AD; (4) LAPC-9 AI; (5) LNCaP; (6) PC-3; (7) DU145; (8) TsuPr1;(9) LAPC-4 CL; (10) HT1197; (11) SCaBER; (12) UM-UC-3; (13) TCCSUP; (14)J82; (15) 5637; (16) 293T; (17) RD-ES; (18) PANC-1; (19) BxPC-3; (20)HPAC; (21) Capan-1; (22) SK-CO-1; (23) CaCo-2; (24) LoVo; (25) T84; (26)Colo-205; (27) KCL 22; (28) PFSK-1; (29) T98G; (30) SK-ES-1; (31) HOS;(32) U2-OS; (33) RD-ES; (34) CALU-1; (35) A427; (36) NCI-H82; (37)NCI-H146; (38) 769-P; (39) A498; (40) CAKI-1; (41) SW839; (42) BT20;(43) CAMA-1; (44) DU4475; (45) MCF-7; (46) MDA-MB-435s; (47) NTERRA-2;(48) NCCIT; (49) TERA-1; (50) TERA-2; (51) A431; (52) HeLa; (53)OV-1063; (54) PA-1; (55) SW626; (56) CAOV-3.

FIG. 7. shows the northern blot analysis of 103P2D6 expression invarious LAPC-4 AD xenografts, including subcutaneously grown xenografts(sc), intratibially grown xenografts (it), and xenografts grown withinhuman bone explants (LAPC-4 AD²) in SCID mice. Lanes represent (1)LAPC-4 AD sc; (2) LAPC-4 AD sc; (3) LAPC-4 AD sc; (4) LAPC-4 AD it; (5)LAPC-4 AD it; (6) LAPC-4 AD it; (7) LAPC-4 AD².

FIG. 8. shows a northern blot analysis of 103P2D6 expression in 9 weekold fetal tissues, including 6 organs and whole embryo. Lanes represent(1) brain; (2) heart; (3) kidney; (4) liver; (5) lung; (6) muscle; (7)whole embryo.

FIG. 9 shows a RT-PCR Expression analysis of 103P2D6. cDNAs generatedusing pools of tissues from multiple normal and cancer tissues werenormalized using beta-actin primers and used to study the expression of103P2D6. Aliquots of the RT-PCR mix after 26 (upper portion of thisfigure) and 30 cycles (lower portion of this figure) were run on theagarose gel to allow semi-quantitative evaluation of the levels ofexpression between samples. The first strand cDNAs in the various lanesof this figure are as follows: Lane 1 (VP-1) contains liver, lung, andkidney first strand cDNA from normal tissues; lane 2 (VP-2) stomach,spleen, and pancreas from normal tissues; lane 3 (xenograft tissue pool)LAPC4AD, LAPC4AI, LAPC9AD, and LAPC9AI; lane 4 is normal prostate tissuepool; lane 5 is prostate cancer tissue pool; lane 6 is bladder cancertissue pool; lane 7 is kidney cancer tissue pool; lane 8 is colon cancertissue pool; lane 9 is from a lung cancer patient; and lane 10 is waterblank.

FIG. 10. shows the results of RT-PCR analysis of 103P2D6 expression inpatient-derived cancers. Lane 1 contains a sample from normal prostate;lane 2 from normal kidney; lane 3 from a prostate tumor pool; lane 4from a kidney tumor pool; lane 5 from a bladder tumor pool; lane 6 fromHeLa cells; and for lane 7 water was used.

FIG. 11A–C. shows expression of 103P2D6 in pancreatic, colon, andprostate cancer cell lines. In panels A and B, cell lysates (˜25 μg)from the indicated cell lines were separated by SDS-PAGE and subjectedto Western blot analysis using an anti-103P2D6 pAb. Indicated with anarrow is a strong anti-103P2D6 pAb immunoreactive band of approximately60 kD present in the pancreatic cancer cell lines HPAC and Bx PC-3, thecolon cancer cell line CaCo-2, and a less intense band in LAPC9 prostatecancer cells indicative of endogenous 103P2D6 protein expression. Alsoindicated with an arrow is the 85 kD immunoreactive band present in 293Tcells transfected with V5-His tagged 103P2D6 cDNA. In panel C, Bx PC-3pancreatic cancer cells were stained with anti-103P2D6 pAb (10 μml) orcontrol rabbit IgG Ab and subjected to flow cytometric analysisfollowing incubation with anti-rabbit IgG-FITC conjugated secondary Ab.Bx PC-3 cells stained with the anti-103P2D6 pAb exhibited a fluorescenceshift compared to the cells stained with control rabbit IgG, indicatingcell surface expression of 103P2D6.

FIG. 12A–B shows expression of 103P2D6 protein in 293T cells. For thedata in panel A, 293T cells were transiently transfected with eitherpCDNA 3.1 V5-His 103P2D6 plasmid or with empty control vector andharvested 2 days later. Cells were lysed in SDS-PAGE sample buffer andlysates were separated by SDS-PAGE gel and transferred tonitrocellulose. Western blotting was carried out with an anti-103P2D6rabbit pAb (2 μg/ml) raised against a peptide encoding amino acids163–176 in the 103P2D6 extracellular domain. Anti-103P2D6 immunoreactivebands were detected by incubation with anti-rabbit-HRP conjugatedsecondary Ab and developed using enhanced chemiluminescence and exposureto autoradiographic film. Indicated by arrow is a specific anti-103P2D6immunoreactive band of approximately 85 kD in 103P2D6-transfected cellsbut not in control cells. For the data in panel B, 103P2D6 transfectedand vector transfected cells were stained with 10 μml of anti-103P2D6pAb and subjected to flow cytometry following incubation withanti-rabbit-FITC conjugated secondary Ab. Shown is a fluorescent shiftin 103P2D6-transfected cells compared to the vector transfected cells,indicating cell surface expression of 103P2D6 protein.

FIG. 13 shows the expression of 103P2D6 as assayed in a panel of humancancers (T) and their respective matched normal tissues (N) on RNA dotblots. 103P2D6 expression was seen in cancers of the kidney, breast,prostate, uterus, ovary, cervix, colon, stomach and rectum 103P2D6 wasalso found to be highly expressed in the two human cancer cell lines,the CML line K562 and the colorectal carcinoma SW480. The expressiondetected in normal adjacent tissues (isolated from diseased tissues) butnot in normal tissues, isolated from healthy donors, indicates thatthese tissues are not fully normal and that 103P2D6 may be expressed inearly stage tumors and that it has utility as a diagnostic marker.Cancer cell lines are, from left to right, HeLa (cervical carcinoma);Daudi (Burkitt's lymphoma); K562 (CML); HL-60 (PML); G361 (melanoma);A549 (lung carcinoma); MOLT-4 (lymphoblastic leuk.); SW480 (colorectalcarcinoma); Raji (Burkitt's lymphoma).

FIG. 14 shows data where RNA was isolated from prostate tumors (T) andtheir adjacent normal tissues (N) obtained from the following prostatecancer patients (Pt); patient 1, Gleason score 4+5; patient 2, Gleasonscore 3+4; and, patient 3, Gleason score 4+3. NP=normal prostate.Northern analysis was performed using 10 μg of total RNA for eachsample. Expression of 103P2D6 was seen in all three tumor samples testedand their respective normal tissues.

FIG. 15 provides data for Northern experiments where RNA was isolatedfrom kidney tumors (T) and their adjacent normal tissues (N) obtainedfrom the following kidney cancer patients: Patient 1-Papillary Type,Stage I, Grade 2/4; Patient 2-Invasive papillary carcinoma, Grade 2/4;Patient 3—Clear cell type Grade 1/3, focally 2/3; Patient 4—Clear celltype, stage III, Grade 2/4; Patient 5—Clear cell type, stage III, Grade3/4; Patient 6—Clear cell type, stage III, Grade 3/4; Patient 7—Clearcell type, Grade III. CL=Cell lines (from left to right): 769-P, A498,SW839; NK=Normal kidney; N=Normal adjacent tissue; T=Tumor. The Northernanalysis was performed using 10 μg of total RNA for each sample.Elevated expression of 103P2D6 was observed in kidney tumors and normaladjacent tissues isolated from kidney cancer patients as compared tonormal kidney.

FIG. 16 shows the results of Northern analysis where RNA was isolatedfrom bladder cancers and adjacent normal tissue obtained from bladdercancer patients. The Northern analysis was performed using 10 μg oftotal RNA for each sample. Expression of 103P2D6 was seen in bladdertumor but not in normal adjacent tissue. N_(at)=Normal adjacent tissue;T=Tumor.

DETAILED DESCRIPTION OF THE INVENTION I.) Defintions

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

As used herein, the terms “advanced prostate cancer”, “locally advancedprostate cancer”, “advanced disease” and “locally advanced disease” meanprostate cancers that have extended through the prostate capsule, andare meant to include stage C disease under the American UrologicalAssociation (AUA) system, stage C1–C2 disease under the Whitmore-Jewettsystem, and stage T3–T4 and N+ disease under the TNM (tumor, node,metastasis) system. In general, surgery is not recommended for patientswith locally advanced disease, and these patients have substantiallyless favorable outcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence 103P2D6 (either by removing the underlying glycosylationsite or by deleting the glycosylation by chemical and/or enzymaticmeans), and/or adding one or more glycosylation sites that are notpresent in the native sequence 103P2D6. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present.

The term “analog” refers to a molecule that is structurally similar orshares similar or corresponding attributes with another molecule (e.g. a103P2D6-related protein). For example an analog of the 103P2D6 proteincan be specifically bound by an antibody or T cell that specificallybinds to 103P2D6.

The term “antibody” is used in the broadest sense. Therefore an“antibody” can be naturally occurring or man-made such as monoclonalantibodies produced by conventional hybridoma technology. Anti-103P2D6antibodies comprise monoclonal and polyclonal antibodies as well asfragments containing the antigen-binding domain and/or one or morecomplementarity determining regions of these antibodies.

As used herein, an “antibody fragment” is defined as at least a portionof the variable region of the immunoglobulin molecule that binds to itstarget, i.e., the antigen-binding region. In one embodiment itspecifically covers single anti-103P2D6 antibodies and clones thereof(including agonist, antagonist and neutralizing antibodies) andanti-103P2D6 antibody compositions with polyepitopic specificity.

The term “codon optimized sequences” refers to nucleotide sequences thathave been optimized for a particular host species by replacing anycodons having a usage frequency of less than about 20%. Nucleotidesequences that have been optimized for expression in a given hostspecies by elimination of spurious polyadenylation sequences,elimination of exon/intron splicing signals, elimination oftransposon-like repeats and/or optimization of GC content in addition tocodon optimization are referred to herein as an “expression enhancedsequences.”

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopeschemotherapeutic agents, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof. Examples ofcytotoxic agents include, but are not limited to maytansinoids, ytrium,bismuth ricin, ricin A-chain, doxorubicin, daunorubicin, taxol, ethidiumbromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin,Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin Achain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin,enomycin, curicin, crotin, calicheamicin, sapaonaria officinalisinhibitor, and glucocorticoid and other chemotherapeutic agents, as wellas radioisotopes such as At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³² and radioactive isotopes of Lu. Antibodies may also beconjugated to a anti-cancer pro-drug activating enzyme capable ofconverting the pro-drug to its active form.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

As used herein, the terms “hybridize”, “hybridizing”, “hybridizes” andthe like, used in the context of polynucleotides, are meant to refer toconventional hybridization conditions, preferably such as hybridizationin 50% formamide/6×SSC/0.1% SDS/100 μ/ml ssDNA, in which temperaturesfor hybridization are above 37 degrees C. and temperatures for washingin 0.1×SSC/0.1% SDS are above 55 degrees C.

As used herein, a polynucleotide is said to be “isolated” when it issubstantially separated from contaminant polynucleotides that correspondor are complementary to genes other than the 103P2D6 gene or that encodepolypeptides other than 103P2D6 gene product or fragments thereof. Askilled artisan can readily employ nucleic acid isolation procedures toobtain an isolated 103P2D6 polynucleotide.

As used herein, a protein is said to be “isolated” when physical,mechanical or chemical methods are employed to remove the 103P2D6protein from cellular constituents that are normally associated with theprotein. A skilled artisan can readily employ standard purificationmethods to obtain an isolated 103P2D6 protein. Alternatively, anisolated protein can be prepared by chemical means.

The term “mammal” as used herein refers to any organism classified as amammal, including mice, rats, rabbits, dogs, cats, cows, horses andhumans. In one embodiment of the invention, the mammal is a mouse. Inanother embodiment of the invention, the mammal is a human.

As used herein, the terms “metastatic prostate cancer” and “metastaticdisease” mean prostate cancers that have spread to regional lymph nodesor to distant sites, and are meant to include stage D disease under theAUA system and stage T×N×M+ under the TNM system. As is the case withlocally advanced prostate cancer, surgery is generally not indicated forpatients with metastatic disease, and hormonal (androgen ablation)therapy is a preferred treatment modality. Patients with metastaticprostate cancer eventually develop an androgen-refractory state within12 to 18 months of treatment initiation. Approximately half of theseandrogen-refractory patients die within 6 months after developing thatstatus. The most common site for prostate cancer metastasis is bone.Prostate cancer bone metastases are often osteoblastic rather thanosteolytic (i.e., resulting in net bone formation). Bone metastases arefound most frequently in the spine, followed by the femur, pelvis, ribcage, skull and humerus. Other common sites for metastasis include lymphnodes, lung, liver and brain. Metastatic prostate cancer is typicallydiagnosed by open or laparoscopic pelvic lymphadenectomy, whole bodyradionuclide scans, skeletal radiography, and/or bone lesion biopsy.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the antibodies comprising the population are identical except forpossible naturally occurring mutations that are present in minoramounts.

As used herein “motif” as in biological motif of an 103P2D6-relatedprotein, refers to any set of amino acids forming part of the primarysequence of a protein, either contiguous or capable of being aligned tocertain positions that are generally invariant, that is associated witha particular function (e.g. protein-protein interaction, protein-DNAinteraction, etc) or modification (e.g. that is phosphorylated,glycosylated or amidated), or localization (e.g. secretory sequence,nuclear localization sequence, etc.) or a sequence that is correlatedwith being immunogenic, either humorally or cellularly.

As used herein, the term “polynucleotide” means a polymeric form ofnucleotides of at least 10 bases or base pairs in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide, and is meant to include single and double stranded forms ofDNA and/or RNA. In the art, this term if often used interchangeably with“oligonucleotide”. A polynucleotide can comprise a nucleotide sequencedisclosed herein wherein thymidine (T) (as shown for example in SEQ IDNO: 1) can also be uracil (U); this definition pertains to thedifferences between the chemical structures of DNA and RNA, inparticular the observation that one of the four major bases in RNA isuracil (U) instead of thymidine (T).

As used herein, the term “polypeptide” means a polymer of at least about4, 5, 6, 7, or 8 amino acids. Throughout the specification, standardthree letter or single letter designations for amino acids are used. Inthe art, this term is often used interchangeably with “peptide” or“protein”.

As used herein, a “recombinant” DNA or RNA molecule is a DNA or RNAmolecule that has been subjected to molecular manipulation in vitro.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured nucleic acidsequences to reanneal when complementary strands are present in anenvironment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature that can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, are identified by, but not limited to, those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (PH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium. citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C. “Moderately stringent conditions” are described by, but not limitedto, those in Sambrook et al., Molecular Cloning: A Laboratory Manual,New York: Cold Spring Harbor Press, 1989, and include the use of washingsolution and hybridization conditions (e.g., temperature, ionic strengthand % SDS) less stringent than those described above. An example ofmoderately stringent conditions is overnight incubation at 37° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37–50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

A “transgenic animal” (e.g., a mouse or rat) is an animal having cellsthat contain a transgene, which transgene was introduced into the animalor an ancestor of the animal at a prenatal, e.g., an embryonic stage. A“transgene” is a DNA that is integrated into the genome of a cell fromwhich a transgenic animal develops.

The term “variant” refers to a molecule that exhibits a variation from adescribed type or norm, such as a protein that has one or more differentamino acid residues in the corresponding position(s) of a specificallydescribed protein (e.g. the 103P2D6 protein shown in FIG. 2 and FIG. 3).An analog is an example of a variant protein.

As used herein, the 103P2D6-related gene and 103P2D6-related proteinincludes the 103P2D6 genes and proteins specifically described herein,as well as structurally and/or functionally similar variants or analogof the foregoing. 103P2D6 peptide analogs generally share at least about50%, 60%, 70%, 80%, 90% or more amino acid homology (using BLASTcriteria). 103P2D6 nucleotide analogs preferably share 50%, 60%, 70%,80%, 90% or more nucleic acid homology (using BLAST criteria). In someembodiments, however, lower homology is preferred so as to selectpreferred residues in view of species-specific codon preferences foroptimized protein expression and production and/orimmunogenicity-modulated peptide epitopes tailored to a particulartarget population, e.g. HLA type, as is appreciated by those skilled inthe art.

The 103P2D6-related proteins of the invention include those specificallyidentified herein, as well as allelic variants, conservativesubstitution variants, analogs and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined herein or readily available in the art.Fusion proteins that combine parts of different 103P2D6 proteins orfragments thereof, as well as fusion proteins of a 103P2D6 protein and aheterologous polypeptide are also included. Such 103P2D6 proteins arecollectively referred to as the 103P2D6-related proteins, the proteinsof the invention, or 103P2D6. As used herein, the term “103P2D6-relatedprotein” refers to a polypeptide fragment or an 103P2D6 protein sequenceof 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, or more than 25 amino acids.

II.) Properties of 103P2D6

As disclosed herein, 103P2D6 exhibits specific properties that areanalogous to those found in a family of molecules whose polynucleotides,polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells(HTL) and anti-polypeptide antibodies are used in well known diagnosticassays that examine conditions associated with dysregulated cell growthsuch as cancer, in particular prostate cancer (see, e.g., both itshighly specific pattern of tissue expression as well as itsoverexpression in prostate cancers as described for example in Example3). The best-known member of this class is PSA, the archetypal markerthat has been used by medical practitioners for years to identify andmonitor the presence of prostate cancer (see, e.g., Merrill et al., J.Urol. 163(2): 503–5120 (2000); Polascik et al., J. Urol.Aug;162(2):293–306 (1999) and Fortier et al., J. Nat. Cancer Inst.91(19): 1635–1640(1999)). A variety of other diagnostic markers are alsoused in this context including p53 and K-ras (see, e.g., Tulchinsky etal., Int J Mol Med July 1999;4(l):99–102 and Minimoto et al., CancerDetect Prev 2000;24(l):1–12). Therefore, this disclosure of the 103P2D6polynucleotides and polypeptides (as well as the 103P2D6 polynucleotideprobes and anti-103P2D6 antibodies used to identify the presence ofthese molecules) and their properties allows skilled artisans to utilizethese molecules in methods that are analogous to those used, forexample, in a variety of diagnostic assays directed to examiningconditions associated with cancer.

Typical embodiments of diagnostic methods that utilize the 103P2D6polynucleotides, polypeptides, reactive T cells and antibodies areanalogous to those methods from well-established diagnostic assays thatemploy, e.g., PSA polynucleotides, polypeptides, reactive T cells andantibodies. For example, just as PSA polynucleotides are used as probes(for example in Northern analysis, see, e.g., Sharief et al., Biochem.Mol. Biol. Int. 33(3):567–74(1994)) and primers (for example in PCRanalysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189–1190 (2000))to observe the presence and/or the level of PSA mRNAs in methods ofmonitoring PSA overexpression or the metastasis of prostate cancers, the103P2D6 polynucleotides described herein can be utilized in the same wayto detect 103P2D6 overexpression or the metastasis of prostate and othercancers expressing this gene. Alternatively, just as PSA polypeptidesare used to generate antibodies specific for PSA which can then be usedto observe the presence and/or the level of PSA proteins in methods tomonitor PSA protein overexpression (see, e.g., Stephan et al., Urology55(4):560–3 (2000)) or the metastasis of prostate cells (see, e.g.,Alanen et al., Pathol. Res. Pract. 192(3):233–7 (1996)), the 103P2D6polypeptides described herein can be utilized to generate antibodies foruse in detecting 103P2D6 overexpression or the metastasis of prostatecells and cells of other cancers expressing this gene.

Specifically, because metastases involves the movement of cancer cellsfrom an organ of origin (such as the lung or prostate gland etc.) to adifferent area of the body (such as a lymph node), assays which examinea biological sample for the presence of cells expressing 103P2D6polynucleotides and/or polypeptides can be used to provide evidence ofmetastasis. For example, when a biological sample from tissue that doesnot normally contain 103P2D6-expressing cells (lymph node) is found tocontain 103P2D6-expressing cells such as the 103P2D6 expression seen inLAPC4 and LAPC9, xenografts isolated from lymph node and bonemetastasis, respectively, this finding is indicative of metastasis.

Alternatively 103P2D6 polynucleotides and/or polypeptides can be used toprovide evidence of cancer, for example, when cells in a biologicalsample that do not normally express 103P2D6 or express 103P2D6 at adifferent level are found to express 103P2D6 or have an increasedexpression of 103P2D6 (see, e.g., the 103P2D6 expression in kidney, lungand colon cancer cells and in patient samples etc. shown in FIGS. 4–10).In such assays, artisans may further wish to generate supplementaryevidence of metastasis by testing the biological sample for the presenceof a second tissue restricted marker (in addition to 103P2D6) such asPSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233–237 (1996)).

Just as PSA polynucleotide fragments and polynucleotide variants areemployed by skilled artisans for use in methods of monitoring PSA,103P2D6 polynucleotide fragments and polynucleotide variants are used inan analogous manner. In particular, typical PSA polynucleotides used inmethods of monitoring PSA are probes or primers that consist offragments of the PSA cDNA sequence. Illustrating this, primers used toPCR amplify a PSA polynucleotide must include less than the whole PSAsequence to function in the polymerase chain reaction. In the context ofsuch PCR reactions, skilled artisans generally create a variety ofdifferent polynucleotide fragments that can be used as primers in orderto amplify different portions of a polynucleotide of interest or tooptimize amplification reactions (see, e.g., Caetano-Anolles, G.Biotechniques 25(3): 472–476, 478–480 (1998); Robertson et al., MethodsMol. Biol. 98:121–154 (1998)). An additional illustration of the use ofsuch fragments is provided in Example 3, where a 103P2D6 polynucleotidefragment is used as a probe to show the expression of 103P2D6 RNAs incancer cells. In addition, variant polynucleotide sequences aretypically used as primers and probes for the corresponding mRNAs in PCRand Northern analyses (see, e.g., Sawai et al., Fetal Diagn. Ther.November-December 1996;11(6):407–13 and Current Protocols In MolecularBiology, Volume 2, Unit 2, Frederick M. Ausubul et al. eds., 1995)).Polynucleotide fragments and variants are useful in this context wherethey are capable of binding to a target polynucleotide sequence (e.g.the 103P2D6 polynucleotide shown in SEQ ID NO: 1) under conditions ofhigh stringency.

Furthermore, PSA polypeptides which contain an epitope that can berecognized by an antibody or T cell that specifically binds to thatepitope are used in methods of monitoring PSA. 103P2D6 polypeptidefragments and polypeptide analogs or variants can also be used in ananalogous manner. This practice of using polypeptide fragments orpolypeptide variants to generate antibodies (such as anti-PSA antibodiesor T cells) is typical in the art with a wide variety of systems such asfusion proteins being used by practitioners (see, e.g., CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubulet al. eds., 1995). In this context, each epitope(s) functions toprovide the architecture with which an antibody or T cell is reactive.Typically, skilled artisans create a variety of different polypeptidefragments that can be used in order to generate immune responsesspecific for different portions of a polypeptide of interest (see, e.g.,U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it maybe preferable to utilize a polypeptide comprising one of the 103P2D6biological motifs discussed herein or available in the art. Polypeptidefragments, variants or analogs are typically useful in this context aslong as they comprise an epitope capable of generating an antibody or Tcell specific for a target polypeptide sequence (e.g. the 103P2D6polypeptide shown in SEQ ID NO: 2).

As shown herein, the 103P2D6 polynucleotides and polypeptides (as wellas the 103P2D6 polynucleotide probes and anti-103P2D6 antibodies or Tcells used to identify the presence of these molecules) exhibit specificproperties that make them useful in diagnosing cancers of the prostate.Diagnostic assays that measure the presence of 103P2D6 gene products, inorder to evaluate the presence or onset of a disease condition describedherein, such as prostate cancer, are used to identify patients forpreventive measures or further monitoring, as has been done sosuccessfully with PSA. Moreover, these materials satisfy a need in theart for molecules having similar or complementary characteristics to PSAin situations where, for example, a definite diagnosis of metastasis ofprostatic origin cannot be made on the basis of a test for PSA alone(see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233–237 (1996)),and consequently, materials such as 103P2D6 polynucleotides andpolypeptides (as well as the 103P2D6 polynucleotide probes andanti-103P2D6 antibodies used to identify the presence of thesemolecules) must be employed to confirm metastases of prostatic origin.

Finally, in addition to their use in diagnostic assays, the 103P2D6polynucleotides disclosed herein have a number of other specificutilities such as their use in the identification of oncogeneticassociated chromosomal abnormalities in 2q34, the chromosomal region towhich the 103P2D6 gene maps (see Example 7 below). Moreover, in additionto their use in diagnostic assays, the 103P2D6-related proteins andpolynucleotides disclosed herein have other utilities such as their usein the forensic analysis of tissues of unknown origin (see, e.g.,Takahama K Forensic Sci Int Jun. 28, 1996;80(1–2): 63–9).

Additionally, 103P2D6-related proteins or polynucleotides of theinvention can be used to treat a pathologic condition characterized bythe over-expression of 103P2D6. For example, the amino acid or nucleicacid sequence of FIG. 2, or fragments thereof, can be used to generatean immune response to the 103P2D6 antigen. Antibodies or other moleculesthat react with 103P2D6 can be used to modulate the function of thismolecule, and thereby provide a therapeutic benefit.

III.) 103P2D6 Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of an 103P2D6 gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding an 103P2D6-related protein and fragments thereof, DNA, RNA,DNA/RNA hybrid, and related molecules, polynucleotides oroligonucleotides complementary to an 103P2D6 gene or mRNA sequence or apart thereof, and polynucleotides or oligonucleotides that hybridize toan 103P2D6 gene, mRNA, or to an 103P2D6 encoding polynucleotide(collectively, “103P2D6 polynucleotides”). In all instances whenreferred to in this section, T can also be U in FIG. 2.

Embodiments of a 103P2D6 polynucleotide include: a 103P2D6polynucleotide having the sequence shown in FIG. 2, the nucleotidesequence of 103P2D6 as shown in FIG. 2, wherein T is U; at least 10contiguous nucleotides of a polynucleotide having the sequence as shownin FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotidehaving the sequence as shown in FIG. 2 where T is U. Further 103P2D6nucleotides comprise, where T can be U:

(a) at least 10 contiguous nucleotides of a polynucleotide having thesequence as shown in FIG. 2, from nucleotide residue number 1 throughnucleotide residue number 804; or,

(b) at least 10 contiguous nucleotides of a polynucleotide having thesequence as shown in FIG. 2, from nucleotide residue number 977 throughnucleotide residue number 1036; or,

(c) at least 10 contiguous nucleotides of a polynucleotide having thesequence as shown in FIG. 2, from nucleotide residue number 1414 throughnucleotide residue number 1815; or

(d) a polynucleotide whose starting base is in the range of 1–804 ofFIG. 2 and whose ending base is in the range of 805–2493 of FIG. 2; or

(e) a polynucleotide whose starting base is in the range of 977–1036 ofFIG. 2 and whose ending base is in the range of 1037–2493 of FIG. 2; or

(f) a polynucleotide whose starting base is in the range of 1414–1815 ofFIG. 2 and whose ending base is in the range of 1816–2493 of FIG. 2; or

(g) a polynucleotide whose starting base is in the range of 805–976 ofFIG. 2 and whose ending base is in the range of 977–2493 of FIG. 2; or

(h) a polynucleotide whose starting base is in the range of 805–2493 ofFIG. 2 and whose ending base is in the range of 2494–4727 of FIG. 2; or

(i) a polynucleotide of (d–g) that is at least 10 nucleotide bases inlength; or

(j) a polynucleotide that selectively hybridizes under stringentconditions to a polynucleotide of (a)–(g);

wherein a range is understood to specifically disclose all whole unitpositions thereof. Moreover, a peptide that is encoded by any of theforegoing is also within the scope of the invention.

Also within the scope of the invention is a nucleotide, as well as anypeptide encoded thereby, that starts at any of the following positionsor ranges, and ends at a higher position or range: 1, 804, a range of1–804, 805, a range of 805–976; a range of 805–2493; a range of977–1036, a range of 1037–1413; a range of 1414–1815; a range of1816–2493; a range of 2494–4727; wherein a range as used in this sectionis understood to specifically disclose all whole unit positions thereof.

Another embodiment of the invention comprises a polynucleotide thatencodes a 103P2D6-related protein whose sequence is encoded by the cDNAcontained in the plasmids deposited with American Type CultureCollection as Accession No. PTA-1155 or PTA-1895. Another embodimentcomprises a polynucleotide that hybridizes under stringent hybridizationconditions, to the human 103P2D6 cDNA shown in SEQ ID NO: 1 or to apolynucleotide fragment thereof.

Typical embodiments of the invention disclosed herein include 103P2D6polynucleotides that encode specific portions of the 103P2D6 mRNAsequence (and those which are complementary to such sequences) such asthose that encode the protein and fragments thereof, for example of 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids.

For example, representative embodiments of the invention disclosedherein include: polynucleotides and their encoded peptides themselvesencoding about amino acid 1 to about amino acid 10 of the 103P2D6protein shown in FIG. 2 and FIG. 3, polynucleotides encoding about aminoacid 10 to about amino acid 20 of the 103P2D6 protein shown in FIG. 2and FIG. 3, polynucleotides encoding about amino acid 20 to about aminoacid 30 of the 103P2D6 protein shown in FIG. 2 and FIG. 3,polynucleotides encoding about amino acid 30 to about amino acid 40 ofthe 103P2D6 protein shown in FIG. 2 and FIG. 3, polynucleotides encodingabout amino acid 40 to about amino acid 50 of the 103P2D6 protein shownin FIG. 2 and FIG. 3, polynucleotides encoding about amino acid 50 toabout amino acid 60 of the 103P2D6 protein shown in FIG. 2 and FIG. 3,polynucleotides encoding about amino acid 60 to about amino acid 70 ofthe 103P2D6 protein shown in FIG. 2 and FIG. 3, polynucleotides encodingabout amino acid 70 to about amino acid 80 of the 103P2D6 protein shownin FIG. 2 and FIG. 3, polynucleotides encoding about amino acid 80 toabout amino acid 90 of the 103P2D6 protein shown in FIG. 2 and FIG. 3and polynucleotides encoding about amino acid 90 to about amino acid 100of the 103P2D6 protein shown in FIG. 2 and FIG. 3, in increments ofabout 10 amino acids, ending at amino acid 532. Accordinglypolynucleotides encoding portions of the amino acid sequence (of about10 amino acids), of amino acids 100–532 of the 103P2D6 protein areembodiments of the invention. Wherein it is understood that eachparticular amino acid position discloses that position plus or minusfive amino acid residues.

Polynucleotides encoding relatively long portions of the 103P2D6 proteinare also within the scope of the invention. Additional illustrativeembodiments of the invention disclosed herein include 103P2D6polynucleotide fragments encoding one or more of the biological motifscontained within the 103P2D6 protein sequence, including one or more ofthe motif-bearing subsequences of the 103P2D6 protein set forth in TableXIX. In another embodiment, typical polynucleotide fragments of theinvention encode one or more of the regions of 103P2D6 that exhibithomology to a known molecule. In another embodiment of the invention,typical polynucleotide fragments can encode one or more of the 103P2D6N-glycosylation sites, cAMP and cGMP-dependent protein kinasephosphorylation sites, casein kinase II phosphorylation sites orN-myristoylation site and amidation sites.

III.A.) Uses of 103P2D6 Polynucleotides

III.A.1.) Monitoring of Genetic Abnormalities

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. The human 103P2D6 gene maps to chromosome4p12–p14 as determined using the GeneBridge4 radiation hybrid panel (seeExample 7). For example, because the 103P2D6 gene maps to chromosome4p12–p14, polynucleotides that encode different regions of the 103P2D6protein are used to characterize cytogenetic abnormalities on chromosome4, band p12–p14 that have been identified as being associated withvarious cancers. In particular, a variety of chromosomal abnormalitiesin 4p12–p14 including translocations and deletions have been identifiedas frequent cytogenetic abnormalities in a number of different cancers(see, e.g., Zimonjic, D. B. et al., 1999, Hepatology 29(4):1208–14; Wu,X. et al., 1995, Cancer Res. 55(3):557–61; Arribas, R. et al., 1999,Lab. Invest. 79(2):111–22). Thus, polynucleotides encoding specificregions of the 103P2D6 protein provide new tools that can be used todelineate, with greater precision than previously possible, cytogeneticabnormalities in this region of chromosome 2 that may contribute to themalignant phenotype. In this context, these polynucleotides satisfy aneed in the art for expanding the sensitivity of chromosomal screeningin order to identify more subtle and less common chromosomalabnormalities (see e.g. Evans et al., Am. J. Obstet. Gynecol 171(4):1055–1057 (1994)).

Furthermore, as 103P2D6 was shown to be highly expressed in prostate andother cancers, 103P2D6 polynucleotides are used in methods assessing thestatus of 103P2D6 gene products in normal versus cancerous tissues.Typically, polynucleotides that encode specific regions of the 103P2D6protein are used to assess the presence of perturbations (such asdeletions, insertions, point mutations, or alterations resulting in aloss of an antigen etc.) in specific regions of the 103P2D6 gene, suchas such regions containing one or more motifs. Exemplary assays includeboth RT-PCR assays as well as single-strand conformation polymorphism(SSCP) analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8):369–378 (1999), both of which utilize polynucleotides encoding specificregions of a protein to examine these regions within the protein.

III.A.2.) Antisense Embodiments

Other specifically contemplated nucleic acid related embodiments of theinvention disclosed herein are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone, or including alternative bases, whether derivedfrom natural sources or synthesized, and include molecules capable ofinhibiting the RNA or protein expression of 103P2D6. For example,antisense molecules can be RNAs or other molecules, including peptidenucleic acids (PNAs) or non-nucleic acid molecules such asphosphorothioate derivatives, that specifically bind DNA or RNA in abase pair-dependent manner. A skilled artisan can readily obtain theseclasses of nucleic acid molecules using the 103P2D6 polynucleotides andpolynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,103P2D6. See for example, Jack Cohen, Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1–5(1988). The 103P2D6 antisense oligonucleotides of the present inventioninclude derivatives such as S-oligonucleotides (phosphorothioatederivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhancedcancer cell growth inhibitory action. S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(O-oligo) in which a nonbridging oxygen atom of the phosphate group isreplaced by a sulfur atom. The S-oligos of the present invention can beprepared by treatment of the corresponding O-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transferreagent. See Iyer, R. P. et al, J. Org. Chem. 55:4693–4698 (1990); andIyer, R. P. et al., J. Am. Chem. Soc. 112:1253–1254 (1990). Additional103P2D6 antisense oligonucleotides of the present invention includemorpholino antisense oligonucleotides known in the art (see, e.g.,Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6:169–175).

The 103P2D6 antisense oligonucleotides of the present inventiontypically can be RNA or DNA that is complementary to and stablyhybridizes with the first 100 5′ codons or last 100 3′ codons of the103P2D6 genomic sequence or the corresponding mRNA. Absolutecomplementarity is not required, although high degrees ofcomplementarity are preferred. Use of an oligonucleotide complementaryto this region allows for the selective hybridization to 103P2D6 mRNAand not to mRNA specifying other regulatory subunits of protein kinase.In one embodiment, 103P2D6 antisense oligonucleotides of the presentinvention are 15 to 30-mer fragments of the antisense DNA molecule thathave a sequence that hybridizes to 103P2D6 mRNA. Optionally, 103P2D6antisense oligonucleotide is a 30-mer oligonucleotide that iscomplementary to a region in the first 10 5′ codons or last 10 3′ codonsof 103P2D6. Alternatively, the antisense molecules are modified toemploy ribozymes in the inhibition of 103P2D6 expression, see, e.g., L.A. Couture & D. T. Stinchcomb; Trends Genet 12: 510–515 (1996).

III.A.3.) Primers and Primer Pairs

Further specific embodiments of this nucleotides of the inventioninclude primers and primer pairs, which allow the specific amplificationof polynucleotides of the invention or of any specific parts thereof,and probes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes can be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers are used todetect the presence of a 103P2D6 polynucleotide in a sample and as ameans for detecting a cell expressing a 103P2D6 protein.

Examples of such probes include polypeptides comprising all or part ofthe human 103P2D6 cDNA sequences shown in FIG. 2. Examples of primerpairs capable of specifically amplifying 103P2D6 mRNAs are alsodescribed in the Examples. As will be understood by the skilled artisan,a great many different primers and probes can be prepared based on thesequences provided herein and used effectively to amplify and/or detecta 103P2D6 mRNA.

The 103P2D6 polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their use as probes and primersfor the amplification and/or detection of the 103P2D6 gene(s), mRNA(s),or fragments thereof; as reagents for the diagnosis and/or prognosis ofprostate cancer and other cancers; as coding sequences capable ofdirecting the expression of 103P2D6 polypeptides; as tools formodulating or inhibiting the expression of the 103P2D6 gene(s) and/ortranslation of the 103P2D6 transcript(s); and as therapeutic agents.

III.A.4.) Isolation of 103P2D6-Encoding Nucleic Acid Molecules

The 103P2D6 cDNA sequences described herein enable the isolation ofother polynucleotides encoding 103P2D6 gene product(s), as well as theisolation of polynucleotides encoding 103P2D6 gene product homologs,alternatively spliced isoforms, allelic variants, and mutant forms ofthe 103P2D6 gene product as well as polynucleotides that encode analogsof 103P2D6-related proteins. Various molecular cloning methods that canbe employed to isolate full length cDNAs encoding an 103P2D6 gene arewell known (See, for example, Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2d edition., Cold Spring Harbor Press, New York,1989; Current Protocols in Molecular Biology. Ausubel et al., Eds.,Wiley and Sons, 1995). For example, lambda phage cloning methodologiescan be conveniently employed, using commercially available cloningsystems (e.g., Lambda ZAP Express, Stratagene). Phage clones containing103P2D6 gene cDNAs can be identified by probing with a labeled 103P2D6cDNA or a fragment thereof. For example, in one embodiment, the 103P2D6cDNA (FIG. 2) or a portion thereof can be synthesized and used as aprobe to retrieve overlapping and full-length cDNAs corresponding to a103P2D6 gene. The 103P2D6 gene itself can be isolated by screeninggenomic DNA libraries, bacterial artificial chromosome libraries (BACs),yeast artificial chromosome libraries (YACs), and the like, with 103P2D6DNA probes or primers.

III.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containingan 103P2D6 polynucleotide, fragment, analog or homologue thereof,including but not limited to phages, plasmids, phagemids, cosmids, YACs,BACs, as well as various viral and non-viral vectors well known in theart, and cells transformed or transfected with such recombinant DNA orRNA molecules. Methods for generating such molecules are well known(see, for example, Sambrook et al, 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a 103P2D6 polynucleotide, fragment,analog or homologue thereof within a suitable prokaryotic or eukaryotichost cell. Examples of suitable eukaryotic host cells include a yeastcell, a plant cell, or an animal cell, such as a mammalian cell or aninsect cell (e.g., a baculovirus-infectible cell such as an Sf9 orHighFive cell). Examples of suitable mammalian cells include variousprostate cancer cell lines such as DU145 and TsuPr1, other transfectableor transducible prostate cancer cell lines, primary cells (PrEC), aswell as a number of mammalian cells routinely used for the expression ofrecombinant proteins (e.g., COS, CHO, 293, 293T cells). Moreparticularly, a polynucleotide comprising the coding sequence of 103P2D6or a fragment, analog or homolog thereof can be used to generate 103P2D6proteins or fragments thereof using any number of host-vector systemsroutinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression of103P2D6 proteins or fragments thereof are available, see for example,Sambrook et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using theseexpression vectors, 103P2D6 can be expressed in several prostate cancerand non-prostate cell lines, including for example 293, 293T, rat-1, NIH3T3 and TsuPr1. The host-vector systems of the invention are useful forthe production of a 103P2D6 protein or fragment thereof. Suchhost-vector systems can be employed to study the functional propertiesof 103P2D6 and 103P2D6 mutations or analogs.

Recombinant human 103P2D6 protein or an analog or homolog or fragmentthereof can be produced by mammalian cells transfected with a constructencoding a 103P2D6-related nucleotide. For example, 293T cells can betransfected with an expression plasmid encoding 103P2D6 or fragment,analog or homolog thereof, the 103P2D6 or related protein is expressedin the 293T cells, and the recombinant 103P2D6 protein is isolated usingstandard purification methods (e.g., affinity purification usinganti-103P2D6 antibodies). In another embodiment, a 103P2D6 codingsequence is subcloned into the retroviral vector pSRαMSVtkneo and usedto infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 andrat-1 in order to establish 103P2D6 expressing cell lines. Various otherexpression systems well known in the art can also be employed.Expression constructs encoding a leader peptide joined in frame to the103P2D6 coding sequence can be used for the generation of a secretedform of recombinant 103P2D6 protein.

As discussed herein, redundancy in the genetic code permits variation in103P2D6 gene sequences. In particular, it is known in the art thatspecific host species often have specific codon preferences, and thusone can adapt the disclosed sequence as preferred for a desired host.For example, preferred analog codon sequences typically have rare codons(i.e., codons having a usage frequency of less than about 20% in knownsequences of the desired host) replaced with higher frequency codons.Codon preferences for a specific species are calculated, for example, byutilizing codon usage tables available on the INTERNET such as:http://www.dna.affrc.go.jp/˜nakamura/codon.html.

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that are deleterious to gene expression. The GC content of thesequence is adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Wherepossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. Other useful modifications include the addition of atranslational initiation consensus sequence at the start of the openreading frame, as described in Kozak, Mol. Cell Biol., 9:5073–5080(1989). Skilled artisans understand that the general rule thateukaryotic ribosomes initiate translation exclusively at the 5′ proximalAUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS92(7): 2662–2666, (1995) and Kozak NAR 15(20): 8125–8148 (1987)).

IV.) 103P2D6-Related Proteins

Another aspect of the present invention provides 103P2D6-relatedproteins. Specific embodiments of 103P2D6 proteins comprise apolypeptide having all or part of the amino acid sequence of human103P2D6 as shown in FIG. 2. Alternatively, embodiments of 103P2D6proteins comprise variant, homolog or analog polypeptides that havealterations in the amino acid sequence of 103P2D6 shown in FIG. 2.

In general, naturally occurring allelic variants of human 103P2D6 sharea high degree of structural identity and homology (e.g., 90% or morehomology). Typically, allelic variants of the 103P2D6 protein containconservative amino acid substitutions within the 103P2D6 sequencesdescribed herein or contain a substitution of an amino acid from acorresponding position in a homologue of 103P2D6. One class of 103P2D6allelic variants are proteins that share a high degree of homology withat least a small region of a particular 103P2D6 amino acid sequence, butfurther contain a radical departure from the sequence, such as anon-conservative substitution, truncation, insertion or frame shift. Incomparisons of protein sequences, the terms, similarity, identity, andhomology each have a distinct meaning as appreciated in the field ofgenetics. Moreover, orthology and paralogy can be important conceptsdescribing the relationship of members of a given protein family in oneorganism to the members of the same family in other organisms.

Amino acid abbreviations are provided in Table II. Conservative aminoacid substitutions can frequently be made in a protein without alteringeither the conformation or the function of the protein. Such changesinclude substituting any of isoleucine (I), valine (V), and leucine (L)for any other of these hydrophobic amino acids; aspartic acid (D) forglutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) andvice versa; and serine (S) for threonine (T) and vice versa. Othersubstitutions can also be considered conservative, depending on theenvironment of the particular amino acid and its role in thethree-dimensional structure of the protein. For example, glycine (G) andalanine (A) can frequently be interchangeable, as can alanine (A) andvaline (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments (see, e.g. Table III herein;pages 13–15 “Biochemistry” 2^(nd) ED. Lubert Stryer ed (StanfordUniversity); Henikoff et al., PNAS 1992 Vol. 89 10915–10919; Lei et al.,J Biol Chem May 19, 1995; 270(20):11882–6).

Embodiments of the invention disclosed herein include a wide variety ofart-accepted variants or analogs of 103P2D6 proteins such aspolypeptides having amino acid insertions, deletions and substitutions.103P2D6 variants can be made using methods known in the art such assite-directed mutagenesis, alanine scanning, and PCR mutagenesis.Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331(1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassettemutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selectionmutagenesis (Wells et al., Philos. Trans. R., Soc. London Ser A, 317:415(1986)) or other known techniques can be performed on the cloned DNA toproduce the 103P2D6 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence that is involved in aspecific biological activity such as a protein-protein interaction.Among the preferred scanning amino acids are relatively small, neutralamino acids. Such amino acids include alanine, glycine, serine, andcysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main-chain conformation of the variant.Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia,J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yieldadequate amounts of variant, an isosteric amino acid can be used.

As defined herein, 103P2D6 variants, analogs or homologs, have thedistinguishing attribute of having at least one epitope that is “crossreactive” with a 103P2D6 protein having the amino acid sequence of SEQID NO: 2. As used in this sentence, “cross reactive” means that anantibody or T cell that specifically binds to an 103P2D6 variant alsospecifically binds to the 103P2D6 protein having the amino acid sequenceof SEQ ID NO: 2. A polypeptide ceases to be a variant of the proteinshown in SEQ ID NO: 2 when it no longer contains any epitope capable ofbeing recognized by an antibody or T cell that specifically binds to the103P2D6 protein. Those skilled in the art understand that antibodiesthat recognize proteins bind to epitopes of varying size, and a groupingof the order of about four or five amino acids, contiguous or not, isregarded as a typical number of amino acids in a minimal epitope. See,e.g., Nair et al., J. Immunol 2000 165(12): 6949–6955; Hebbes et al.,Mol Immunol (1989) 26(9):865–73; Schwartz et al., J Immunol (1985)135(4):2598–608.

Another class of 103P2D6-related protein variants share 70%, 75%, 80%,85% or 90% or more similarity with the amino acid sequence of SEQ ID NO:2 or a fragment thereof. Another specific class of 103P2D6 proteinvariants or analogs comprise one or more of the 103P2D6 biologicalmotifs described herein or presently known in the art. Thus, encompassedby the present invention are analogs of 103P2D6 fragments (nucleic oramino acid) that have altered function al (e.g. immunogenic) propertiesrelative to the starting fragment. It is to be appreciated that motifsnow or which become part of the art are to be applied to the nucleic oramino acid sequences of FIG. 2.

As discussed herein, embodiments of the claimed invention includepolypeptides containing less than the 532 amino acid sequence of the103P2D6 protein shown in FIG. 2. For example, representative embodimentsof the invention, comprise peptides/proteins having any 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of the 103P2D6protein shown in FIG. 2 and FIG. 3.

Moreover, representative embodiments of the invention disclosed hereininclude polypeptides consisting of about amino acid 1 to about aminoacid 10 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, polypeptidesconsisting of about amino acid 10 to about amino acid 20 of the 103P2D6protein shown in FIG. 2 and FIG. 3, polypeptides consisting of aboutamino acid 20 to about amino acid 30 of the 103P2D6 protein shown inFIG. 2 and FIG. 3, polypeptides consisting of about amino acid 30 toabout amino acid 40 of the 103P2D6 protein shown in FIG. 2 and FIG. 3,polypeptides consisting of about amino acid 40 to about amino acid 50 ofthe 103P2D6 protein shown in FIG. 2 and FIG. 3, polypeptides consistingof about amino acid 50 to about amino acid 60 of the 103P2D6 proteinshown in FIG. 2 and FIG. 3, polypeptides consisting of about amino acid60 to about amino acid 70 of the 20 103P2D6 protein shown in FIG. 2 andFIG. 3, polypeptides consisting of about amino acid 70 to about aminoacid 80 of the 103P2D6 protein shown in FIG. 2 and FIG. 3, polypeptidesconsisting of about amino acid 80 to about amino acid 90 of the 103P2D6protein shown in FIG. 2 and FIG. 3 and polypeptides consisting of aboutamino acid 90 to about amino acid 100 of the 103P2D6 protein shown inFIG. 2 and FIG. 3, etc. throughout the entirety of the 103P2D6 sequence.Further, this definition defines polypeptides consisting of 10 aminoacid stretches of the amino acid sequence of amino acids 100–532 of the103P2D6 protein. Moreover, polypeptides consisting of about amino acid 1(or 20 or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150etc.) of the 103P2D6 protein shown in FIG. 2 and FIG. 3 are embodimentsof the invention. It is to be appreciated that the starting and stoppingpositions in this paragraph refer to the specified position as well asthat position plus or minus 5 residues.

103P2D6-related proteins are generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the art.Alternatively, recombinant methods can be used to generate nucleic acidmolecules that encode a 103P2D6-related protein. In one embodiment,nucleic acid molecules provide a means to generate defined fragments ofthe 103P2D6 protein (or variants, homologs or analogs thereof).

IV.A.) Motif-bearing Protein Embodiments

Additional illustrative embodiments of the invention disclosed hereininclude 103P2D6 polypeptides comprising the amino acid residues of oneor more of the biological motifs contained within the 103P2D6polypeptide sequence set forth in FIG. 2 or FIG. 3. Various motifs areknown in the art, and a protein can be evaluated for the presence ofsuch motifs by a number of publicly available sites (see, e.g.:http://pfam.wustl.edu/;http://searchlauncher.bcm.tmc.edu.seg-search/struc-predict.htmlhttp://psort.ims.u-tokyo.ac.jp/; http://www.cbs.dtu.dk/;http://www.ebi.ac.uk/interpro/scan.html;http://www.expasy.ch/tools/scnpsit1.html; Epimatrix™ and Epimer™, BrownUniversity, http://www.brown.edu/Research/TB-HIVLab/epimatrix/epimatrix.html; and BIMAS, http://bimas.dcrt.nih.gov/.).

Motif bearing subsequences of the 103P2D6 protein are set forth andidentified in Table XIX.

Table XX sets forth several frequently occurring motifs based on pfamsearches (http://pfam.wustl.edu/). The columns of Table XX list (1)motif name abbreviation, (2) percent identity found amongst thedifferent member of the motif family, (3) motif name or description and(4) most common function; location information is included if the motifis relevant for location.

Polypeptides comprising one or more of the 103P2D6 motifs discussedabove are useful in elucidating the specific characteristics of amalignant phenotype in view of the observation that the 103P2D6 motifsdiscussed above are associated with growth dysregulation and because103P2D6 is overexpressed in certain cancers (See, e.g., Table I). Caseinkinase II, cAMP and cCMP-dependent protein kinase, and Protein Kinase C,for example, are enzymes known to be associated with the development ofthe malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2):165–174 (1998); Gaiddon et al., Endocrinology 136(10): 4331–4338 (1995);Hall et al., Nucleic Acids Research 24(6) 1119–1126 (1996); Peterziel etal., Oncogene 18(46): 6322–6329 (1999) and O'Brian, Oncol. Rep. 5(2):305–309 (1998)). Moreover, both glycosylation and myristoylation areprotein modifications also associated with cancer and cancer progression(see e.g. Dennis et al., Biochem Biophys. Acta 1473(1):21–34 (1999);Raju et al., Exp. Cell Res. 235(1): 145–154 (1997)). Amidation isanother protein modification also associated with cancer and cancerprogression (see e.g. Treston et al., J. Natl. Cancer Inst. Monogr.(13): 169–175 (1992)).

In another embodiment, proteins of the invention comprise one or more ofthe immunoreactive epitopes identified in accordance with art-acceptedmethods, such as the peptides set forth in Tables V–XVIII. CTL epitopescan be determined using specific algorithms to identify peptides withinan 103P2D6 protein that are capable of optimally binding to specifiedHLA alleles (e.g., Table IV (A) and Table IV (B); Epimatrix™ andEpimer™, Brown University, http://www.brown.edu/Research/TB-HIVLab/epimatrix/epimatrix.html; and BIMAS, http://bimas.dcrt.nih.gov/.Moreover, processes for identifying peptides that have sufficientbinding affinity for HLA molecules and which are correlated with beingimmunogenic epitopes, are well known in the art, and are carried outwithout undue experimentation. In addition, processes for identifyingpeptides that are immunogenic epitopes, are well known in the art, andare carried out without undue experimentation either in vitro or invivo.

Also known in the art are principles for creating analogs of suchepitopes in order to modulate immunogenicity. For example, one beginswith an epitope that bears a CTL or HTL motif (see, e.g., the HLA ClassI motifs or Table IV (A) and the HTL motif of Table IV (B)). The epitopeis analoged by substituting out an amino acid at one of the specifiedpositions, and replacing it with another amino acid specified for thatposition.

A variety of references reflect the art regarding the identification andgeneration of epitopes in a protein of interest as well as analogsthereof. See, for example, WO 9733602 to Chestnut et al.; Sette,Immunogenetics 1999 50(3–4): 201–212; Sette et al., J. Immunol. 2001166(2): 1389–1397; Sidney et al., Hum. Immunol. 1997 58(1): 12–20; Kondoet al., Immunogenetics 1997 45(4): 249–258; Sidney et al., J. Immunol.1996 157(8): 3480–90; and Falk et al., Nature 351: 290–6 (1991); Hunt etal., Science 255:1261–3 (1992); Parker et al., J. Immunol. 149:3580–7(1992); Parker et al., J. Immunol. 152:163–75 (1994)); Kast et al., 1994152(8): 3904–12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3):266–278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625–1633;Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J.Immunol. 1991 147(8): 2663–2669; Alexander et al., Immunity 1994 1(9):751–761 and Alexander et al., Immunol. Res. 1998 18(2): 79–92.

Related embodiments of the inventions include polypeptides comprisingcombinations of the different motifs set forth in Table XIX, and/or, oneor more of the predicted CTL epitopes of Table V through Table XVIII,and/or, one or more of the T cell binding motifs known in the art.Preferred embodiments contain no insertions, deletions or substitutionseither within the motifs or the intervening sequences of thepolypeptides. In addition, embodiments which include a number of eitherN-terminal and/or C-terminal amino acid residues on either side of thesemotifs may be desirable (to, for example, include a greater portion ofthe polypeptide architecture in which the motif is located). Typicallythe number of N-terminal and/or C-terminal amino acid residues on eitherside of a motif is between about 1 to about 100 amino acid residues,preferably 5 to about 50 amino acid residues.

103P2D6-related proteins are embodied in many forms, preferably inisolated form. A purified 103P2D6 protein molecule will be substantiallyfree of other proteins or molecules that impair the binding of 103P2D6to antibody, T cell or other ligand. The nature and degree of isolationand purification will depend on the intended use. Embodiments of a103P2D6-related proteins include purified 103P2D6-related proteins andfunctional, soluble 103P2D6-related proteins. In one embodiment, afunctional, soluble 103P2D6 protein or fragment thereof retains theability to be bound by antibody, T cell or other ligand.

The invention also provides 103P2D6 proteins comprising biologicallyactive fragments of the 103P2D6 amino acid sequence shown in FIG. 2.Such proteins exhibit properties of the 103P2D6 protein, such as theability to elicit the generation of antibodies that specifically bind anepitope associated with the 103P2D6 protein; to be bound by suchantibodies; to elicit the activation of HTL or CTL; and/or, to berecognized by HTL or CTL.

103P2D6-related polypeptides that contain particularly interestingstructures can be predicted and/or identified using various analyticaltechniques well known in the art, including, for example, the methods ofChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis, or on the basis of immunogenicity. Fragmentsthat contain such structures are particularly useful in generatingsubunit-specific anti-103P2D6 antibodies, or T cells or in identifyingcellular factors that bind to 103P2D6.

CTL epitopes can be determined using specific algorithms to identifypeptides within an 103P2D6 protein that are capable of optimally bindingto specified HLA alleles (e.g., Table IV (A) and Table IV (B);Epimatrix™ and Epimer™, Brown University(http://www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); andBIMAS, http://bimas.dcrt.nih.gov/). Illustrating this, peptide epitopesfrom 103P2D6 that are presented in the context of human MHC class Imolecules HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (TablesV–XVIII). Specifically, the complete amino acid sequence of the 103P2D6protein was entered into the HLA Peptide Motif Search algorithm found inthe Bioinformatics and Molecular Analysis Section (BIMAS) web sitelisted above. The HLA peptide motif search algorithm was developed byDr. Ken Parker based on binding of specific peptide sequences in thegroove of HLA Class I molecules and specifically HLA-A2 (see, e.g., Falket al., Nature 351: 290–6 (1991); Hunt et al., Science 255:1261–3(1992); Parker et al., J. Immunol. 149:3580–7 (1992); Parker et al., J.Immunol. 152:163–75 (1994)). This algorithm allows location and rankingof 8-mer, 9-mer, and 10-mer peptides from a complete protein sequencefor predicted binding to HLA-A2 as well as numerous other HLA Class Imolecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers.For example, for class I HLA-A2, the epitopes preferably contain aleucine (L) or methionine (M) at position 2 and a valine (V) or leucine(L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580–7(1992)). Selected results of 103P2D6 predicted binding peptides areshown in Tables V–XVIII herein. In Tables V–XVIII, the top 50 rankingcandidates, 9-mers and 10-mers, for each family member are shown alongwith their location, the amino acid sequence of each specific peptide,and an estimated binding score. The binding score corresponds to theestimated half-time of dissociation of complexes containing the peptideat 37° C. at pH 6.5. Peptides with the highest binding score arepredicted to be the most tightly bound to HLA Class I on the cellsurface for the greatest period of time and thus represent the bestimmunogenic targets for T-cell recognition.

Actual binding of peptides to an HLA allele can be evaluated bystabilization of HLA expression on the antigen-processing defective cellline T2 (see, e.g., Xue et al., Prostate 30:73–8 (1997) and Peshwa etal., Prostate 36:129–38 (1998)). Immunogenicity of specific peptides canbe evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes(CTL) in the presence of antigen presenting cells such as dendriticcells.

It is to be appreciated that every epitope predicted by the BIMAS site,Epimer™ and Epimatrix™ sites, or specified by the HLA class I or class Imotifs available in the art or which become part of the art such as setforth in Table IV (A) and Table IV (B) are to be “applied” to the103P2D6 protein. As used in this context “applied” means that the103P2D6 protein is evaluated, e.g., visually or by computer-basedpatterns finding methods, as appreciated by those of skill in therelevant art. Every subsequence of the 103P2D6 of 8, 9, 10, or 11 aminoacid residues that bears an HLA Class I motif, or a subsequence of 9 ormore amino acid residues that bear an HLA Class II motif are within thescope of the invention.

IV.B.) Expression of 103P2D6-Related Proteins

In an embodiment described in the examples that follow, 103P2D6 can beconveniently expressed in cells (such as 293T cells) transfected with acommercially available expression vector such as a CMV-driven expressionvector encoding 103P2D6 with a C-terminal 6×His and MYC tag(pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, NashvilleTenn.). The Tag5 vector provides an IgGK secretion signal that can beused to facilitate the production of a secreted 103P2D6 protein intransfected cells. The secreted HIS-tagged 103P2D6 in the culture mediacan be purified, e.g., using a nickel column using standard techniques.

IV.C.) Modifications of 103P2D6-related Proteins

Modifications of 103P2D6-related proteins such as covalent modificationsare included within the scope of this invention. One type of covalentmodification includes reacting targeted amino acid residues of a 103P2D6polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues ofthe 103P2D6. Another type of covalent modification of the 103P2D6polypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of a protein of the invention.Another type of covalent modification of 103P2D6 comprises linking the103P2D6 polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The 103P2D6-related proteins of the present invention can also bemodified to form a chimeric molecule comprising 103P2D6 fused toanother, heterologous polypeptide or amino acid sequence. Such achimeric molecule can be synthesized chemically or recombinantly. Achimeric molecule can have a protein of the invention fused to anothertumor-associated antigen or fragment thereof. Alternatively, a proteinin accordance with the invention can comprise a fusion of fragments ofthe 103P2D6 sequence (amino or nucleic acid) such that a molecule iscreated that is not, through its length, directly homologous to theamino or nucleic acid sequences respectively of FIG. 2. Such a chimericmolecule can comprise multiples of the same subsequence of 103P2D6. Achimeric molecule can comprise a fusion of a 103P2D6-related proteinwith a polyhistidine epitope tag, which provides an epitope to whichimmobilized nickel can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the 103P2D6. In analternative embodiment, the chimeric molecule can comprise a fusion of a103P2D6-related protein with an immunoglobulin or a particular region ofan immunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a 103P2D6 polypeptide in place of at least one variable regionwithin an Ig molecule. In a preferred embodiment, the immunoglobulinfusion includes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3regions of an IgGI molecule. For the production of immunoglobulinfusions see, e.g., U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

IV.D.) Uses of 103P2D6-related Proteins

The proteins of the invention have a number of different specific uses.As 103P2D6 is highly expressed in prostate and other cancers,103P2D6-related proteins are used in methods that assess the status of103P2D6 gene products in normal versus cancerous tissues, therebyelucidating the malignant phenotype. Typically, polypeptides fromspecific regions of the 103P2D6 protein are used to assess the presenceof perturbations (such as deletions, insertions, point mutations etc.)in those regions (such as regions containing one or more motifs).Exemplary assays utilize antibodies or T cells targeting 103P2D6-relatedproteins comprising the amino acid residues of one or more of thebiological motifs contained within the 103P2D6 polypeptide sequence inorder to evaluate the characteristics of this region in normal versuscancerous tissues or to elicit an immune response to the epitope.Alternatively, 103P2D6-related proteins that contain the amino acidresidues of one or more of the biological motifs in the 103P2D6 proteinare used to screen for factors that interact with that region of103P2D6.

103P2D6 protein fragments/subsequences are particularly useful ingenerating and characterizing domain-specific antibodies (e.g.,antibodies recognizing an extracellular or intracelular epitope of an103P2D6 protein), for identifying agents or cellular factors that bindto 103P2D6 or a particular structural domain thereof, and in varioustherapeutic and diagnostic contexts, including but not limited todiagnostic assays, cancer vaccines and methods of preparing suchvaccines.

Proteins encoded by the 103P2D6 genes, or by analogs, homologs orfragments thereof, have a variety of uses, including but not limited togenerating antibodies and in methods for identifying ligands and otheragents and cellular constituents that bind to an 103P2D6 gene product.Antibodies raised against an 103P2D6 protein or fragment thereof areuseful in diagnostic and prognostic assays, and imaging methodologies inthe management of human cancers characterized by expression of 103P2D6protein, such as those listed in Table I. Such antibodies can beexpressed intracellularly and used in methods of treating patients withsuch cancers. 103P2D6-related nucleic acids or proteins are also used ingenerating HTL or CTL responses.

Various immunological assays useful for the detection of 103P2D6proteins are used, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Antibodies can be labeled and used asimmunological imaging reagents capable of detecting 103P2D6-expressingcells (e.g., in radioscintigraphic imaging methods). 103P2D6 proteinsare also particularly useful in generating cancer vaccines, as furtherdescribed herein.

V.) 103P2D6 Antibodies

Another aspect of the invention provides antibodies that bind to103P2D6-related proteins. Preferred antibodies specifically bind to a103P2D6-related protein and do not bind (or bind weakly) to peptides orproteins that are not 103P2D6-related proteins. For example, antibodiesbind 103P2D6 can bind 103P2D6-related proteins such as the homologs oranalogs thereof.

103P2D6 antibodies of the invention are particularly useful in prostatecancer diagnostic and prognostic assays, and imaging methodologies.Similarly, such antibodies are useful in the treatment, diagnosis,and/or prognosis of other cancers, to the extent 103P2D6 is alsoexpressed or overexpressed in these other cancers. Moreover,intracellularly expressed antibodies (e.g., single chain antibodies) aretherapeutically useful in treating cancers in which the expression of103P2D6 is involved, such as advanced or metastatic prostate cancers.

The invention also provides various immunological assays useful for thedetection and quantification of 103P2D6 and mutant 103P2D6-relatedproteins. Such assays can comprise one or more 103P2D6 antibodiescapable of recognizing and binding a 103P2D6-related protein, asappropriate. These assays are performed within various immunologicalassay formats well known in the art, including but not limited tovarious types of radioimmunoassays, enzyme-linked immunosorbent assays(ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cellimmunogenicity assays (inhibitory or stimulatory) as well as majorhistocompatibility complex (MHC) binding assays.

In addition, immunological imaging methods capable of detecting prostatecancer and other cancers expressing 103P2D6 are also provided by theinvention, including but not limited to radioscintigraphic imagingmethods using labeled 103P2D6 antibodies. Such assays are clinicallyuseful in the detection, monitoring, and prognosis of 103P2D6 expressingcancers such as prostate cancer.

103P2D6 antibodies are also used in methods for purifying a103P2D6-related protein and for isolating 103P2D6 homologues and relatedmolecules. For example, a method of purifying a 103P2D6-related proteincomprises incubating an 103P2D6 antibody, which has been coupled to asolid matrix, with a lysate or other solution containing a103P2D6-related protein under conditions that permit the 103P2D6antibody to bind to the 103P2D6-related protein; washing the solidmatrix to eliminate impurities; and eluting the 103P2D6-related proteinfrom the coupled antibody. Other uses of the 103P2D6 antibodies of theinvention include generating anti-idiotypic antibodies that mimic the103P2D6 protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies can be prepared by immunizing a suitablemammalian host using a 103P2D6-related protein, peptide, or fragment, inisolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSHPress, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold SpringHarbor Press, NY (1989)). In addition, fusion proteins of 103P2D6 canalso be used, such as a 103P2D6 GST-fusion protein. In a particularembodiment, a GST fusion protein comprising all or most of the aminoacid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogento generate appropriate antibodies. In another embodiment, a103P2D6-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used(with or without purified 103P2D6-related protein or 103P2D6 expressingcells) to generate an immune response to the encoded immunogen (forreview, see Donnelly et al., 1997, Ann Rev. Immunol. 15: 617–648).

The amino acid sequence of 103P2D6 as shown in FIG. 2 or FIG. 3 can beanalyzed to select specific regions of the 103P2D6 protein forgenerating antibodies. For example, hydrophobicity and hydrophilicityanalyses of the 103P2D6 amino acid sequence are used to identifyhydrophilic regions in the 103P2D6 structure. Regions of the 103P2D6protein that show immunogenic structure, as well as other regions anddomains, can readily be identified using various other methods known inthe art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg,Karplus-Schultz or Jameson-Wolf analysis. Thus, each region identifiedby any of these programs or methods is within the scope of the presentinvention. Methods for the generation of 103P2D6 antibodies are furtherillustrated by way of the examples provided herein. Methods forpreparing a protein or polypeptide for use as an immunogen are wellknown in the art. Also well known in the art are methods for preparingimmunogenic conjugates of a protein with a carrier, such as BSA, KLH orother carrier protein. In some circumstances, direct conjugation using,for example, carbodiimide reagents are used; in other instances linkingreagents such as those supplied by Pierce Chemical Co., Rockford, Ill.,are effective. Administration of a 103P2D6 immunogen is often conductedby injection over a suitable time period and with use of a suitableadjuvant, as is understood in the art. During the immunization schedule,titers of antibodies can be taken to determine adequacy of antibodyformation.

103P2D6 monoclonal antibodies can be produced by various means wellknown in the art. For example, immortalized cell lines that secrete adesired monoclonal antibody are prepared using the standard hybridomatechnology of Kohler and Milstein or modifications that immortalizeantibody-producing B cells, as is generally known. Immortalized celllines that secrete the desired antibodies are screened by immunoassay inwhich the antigen is a 103P2D6-related protein. When the appropriateimmortalized cell culture is identified, the cells can be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced, byrecombinant means. Regions that bind specifically to the desired regionsof the 103P2D6 protein can also be produced in the context of chimericor complementarity determining region (CDR) grafted antibodies ofmultiple species origin. Humanized or human 103P2D6 antibodies can alsobe produced, and are preferred for use in therapeutic contexts. Methodsfor humanizing murine and other non-human antibodies, by substitutingone or more of the non-human antibody CDRs for corresponding humanantibody sequences, are well known (see for example, Jones et al., 1986,Nature 321: 522–525; Riechmnan et al., 1988, Nature 332: 323–327;Verhoeyen et al., 1988, Science 239: 1534–1536). See also, Carter etal., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J.Immunol. 151: 2296.

Methods for producing fully human monoclonal antibodies include phagedisplay and transgenic methods (for review, see Vaughan et al., 1998,Nature Biotechnology 16: 535–539). Fully human 103P2D6 monoclonalantibodies can be generated using cloning technologies employing largehuman Ig gene combinatorial libraries (i.e., phage display) (Griffithsand Hoogenboom, Building an in vitro immune system: human antibodiesfrom phage display libraries. In: Protein Engineering of AntibodyMolecules for Prophylactic and Therapeutic Applications in Man, Clark,M. (Ed.), Nottingham Academic, pp 45–64 (1993); Burton and Barbas, HumanAntibodies from combinatorial libraries. Id., pp 65–82). Fully human103P2D6 monoclonal antibodies can also be produced using transgenic miceengineered to contain human immunoglobulin gene loci as described in PCTPatent Application W098/24893, Kucherlapati and Jakobovits et al.,published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest.Drugs 7(4): 607–614; U.S. Pat. No. 6,162,963 issued Dec. 19, 2000; U.S.Pat. No. 6,150,584 issued 12 Nov. 2000; and, U.S. Pat. No. 6,114,598issued 5 Sep. 2000). This method avoids the in vitro manipulationrequired with phage display technology and efficiently produces highaffinity authentic human antibodies.

Reactivity of 103P2D6 antibodies with an 103P2D6-related protein can beestablished by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,103P2D6-related proteins, 103P2D6-expressing cells or extracts thereof.A 103P2D6 antibody or fragment thereof can be labeled with a detectablemarker or conjugated to a second molecule. Suitable detectable markersinclude, but are not limited to, a radioisotope, a fluorescent compound,a bioluminescent compound, chemiluminescent compound, a metal chelatoror an enzyme. Further, bi-specific antibodies specific for two or more103P2D6 epitopes are generated using methods generally known in the art.Homodimeric antibodies can also be generated by cross-linking techniquesknown in the art (e.g., Wolff et al., Cancer Res. 53: 2560–2565).

VI.) 103P2D6 Transgenic Animals

Nucleic acids that encode a 103P2D6-related protein can also be used togenerate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. In accordance with established techniques, cDNAencoding 103P2D6 can be used to clone genomic DNA that encodes 103P2D6.The cloned genomic sequences can then be used to generate transgenicanimals containing cells that express DNA that encode 103P2D6. Methodsfor generating transgenic animals, particularly animals such as mice orrats, have become conventional in the art and are described, forexample, in U.S. Pat. No. 4,736,866 issued 12 Apr. 1988, and U.S. Pat.No. 4,870,009 issued 26 Sep. 1989. Typically, particular cells would betargeted for 103P2D6 transgene incorporation with tissue-specificenhancers.

Transgenic animals that include a copy of a transgene encoding 103P2D6can be used to examine the effect of increased expression of DNA thatencodes 103P2D6. Such animals can be used as tester animals for reagentsthought to confer protection from, for example, pathological conditionsassociated with its overexpression. In accordance with this aspect ofthe invention, an animal is treated with a reagent and a reducedincidence of a pathological condition, compared to untreated animalsthat bear the transgene, would indicate a potential therapeuticintervention for the pathological condition.

Alternatively, non-human homologues of 103P2D6 can be used to constructa 103P2D6 “knock out” animal that has a defective or altered geneencoding 103P2D6 as a result of homologous recombination between theendogenous gene encoding 103P2D6 and altered genomic DNA encoding103P2D6 introduced into an embryonic cell of the animal. For example,cDNA that encodes 103P2D6 can be used to clone genomic DNA encoding103P2D6 in accordance with established techniques. A portion of thegenomic DNA encoding 103P2D6 can be deleted or replaced with anothergene, such as a gene encoding a selectable marker that can be used tomonitor integration. Typically, several kilobases of unaltered flankingDNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected(see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras (see, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113–152). A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal, and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knock out animals can becharacterized, for example, for their ability to defend against certainpathological conditions or for their development of pathologicalconditions due to absence of the 103P2D6 polypeptide.

VII.) Methods for the Detection of 103P2D6

Another aspect of the present invention relates to methods for detecting103P2D6 polynucleotides and 103P2D6-related proteins, as well as methodsfor identifying a cell that expresses 103P2D6. The expression profile of103P2D6 makes it a diagnostic marker for metastasized disease.Accordingly, the status of 103P2D6 gene products provides informationuseful for predicting a variety of factors including susceptibility toadvanced stage disease, rate of progression, and/or tumoraggressiveness. As discussed in detail herein, the status of 103P2D6gene products in patient samples can be analyzed by a variety protocolsthat are well known in the art including immunohistochemical analysis,the variety of Northern blotting techniques including in situhybridization, RT-PCR analysis (for example on laser capturemicro-dissected samples), Western blot analysis and tissue arrayanalysis.

More particularly, the invention provides assays for the detection of103P2D6 polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable 103P2D6 polynucleotides include, for example, a 103P2D6gene or fragment thereof, 103P2D6 mRNA, alternative splice variant103P2D6 mRNAs, and recombinant DNA or RNA molecules that contain a103P2D6 polynucleotide. A number of methods for amplifying and/ordetecting the presence of 103P2D6 polynucleotides are well known in theart and can be employed in the practice of this aspect of the invention.

In one embodiment, a method for detecting an 103P2D6 mRNA in abiological sample comprises producing cDNA from the sample by reversetranscription using at least one primer; amplifying the cDNA so producedusing an 103P2D6 polynucleotides as sense and antisense primers toamplify 103P2D6 cDNAs therein; and detecting the presence of theamplified 103P2D6 cDNA. Optionally, the sequence of the amplified103P2D6 cDNA can be determined.

In another embodiment, a method of detecting a 103P2D6 gene in abiological sample comprises first isolating genomic DNA from the sample;amplifying the isolated genomic DNA using 103P2D6 polynucleotides assense and antisense primers; and detecting the presence of the amplified103P2D6 gene. Any number of appropriate sense and antisense probecombinations can be designed from the nucleotide sequences provided forthe 103P2D6 (FIG. 2) and used for this purpose.

The invention also provides assays for detecting the presence of an103P2D6 protein in a tissue or other biological sample such as serum,semen, bone, prostate, urine, cell preparations, and the like. Methodsfor detecting a 103P2D6-related protein are also well known and include,for example, immunoprecipitation, immunohistochemical analysis, Westernblot analysis, molecular binding assays, ELISA, ELIFA and the like. Forexample, a method of detecting the presence of a 103P2D6-related proteinin a biological sample comprises first contacting the sample with a103P2D6 antibody, a 103P2D6-reactive fragment thereof, or a recombinantprotein containing an antigen binding region of a 103P2D6 antibody; andthen detecting the binding of 103P2D6-related protein in the sample.

Methods for identifying a cell that expresses 103P2D6 are also withinthe scope of the invention In one embodiment, an assay for identifying acell that expresses a 103P2D6 gene comprises detecting the presence of103P2D6 mRNA in the cell. Methods for the detection of particular mRNAsin cells are well known and include, for example, hybridization assaysusing complementary DNA probes (such as in situ hybridization usinglabeled 103P2D6 riboprobes, Northern blot and related techniques) andvarious nucleic acid amplification assays (such as RT-PCR usingcomplementary primers specific for 103P2D6, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like). Alternatively, an assay for identifying a cell that expressesa 103P2D6 gene comprises detecting the presence of 103P2D6-relatedprotein in the cell or secreted by the cell. Various methods for thedetection of proteins are well known in the art and are employed for thedetection of 103P2D6-related proteins and cells that express103P2D6-related proteins.

103P2D6 expression analysis is also useful as a tool for identifying andevaluating agents that modulate 103P2D6 gene expression. For example,103P2D6 expression is significantly upregulated in prostate cancer, andis expressed in cancers of the tissues listed in Table I. Identificationof a molecule or biological agent that inhibits 103P2D6 expression orover-expression in cancer cells is of therapeutic value. For example,such an agent can be identified by using a screen that quantifies103P2D6 expression by RT-PCR, nucleic acid hybridization or antibodybinding.

VIII.) Methods for Monitoring the Status of 103P2D6-Related Genes andTheir Products

Oncogenesis is known to be a multistep process where cellular growthbecomes progressively dysregulated and cells progress from a normalphysiological state to precancerous and then cancerous states (see,e.g., Alers et al., Lab Invest. 77(5): 437438 (1997) and Isaacs et al.,Cancer Surv. 23: 19–32 (1995)). In this context, examining a biologicalsample for evidence of dysregulated cell growth (such as aberrant103P2D6 expression in cancers) allows for early detection of suchaberrant physiology, before a pathologic state such as cancer hasprogressed to a stage that therapeutic options are more limited and orthe prognosis is worse. In such examinations, the status of 103P2D6 in abiological sample of interest can be compared, for example, to thestatus of 103P2D6 in a corresponding normal sample (e.g. a sample fromthat individual or alternatively another individual that is not effectedby a pathology). An alteration in the status of 103P2D6 in thebiological sample (as compared to the normal sample) provides evidenceof dysregulated cellular growth. In addition to using a biologicalsample that is not effected by a pathology as a normal sample, one canalso use a predetermined normative value such as a predetermined normallevel of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol. Dec9, 1996;376(2):306–14 and U.S. Pat. No. 5,837,501) to compare 103P2D6status in a sample.

The term “status” in this context is used according to its art acceptedmeaning and refers to the condition or state of a gene and its products.Typically, skilled artisans use a number of parameters to evaluate thecondition or state of a gene and its products. These include, but arenot limited to the location of expressed gene products (including thelocation of 103P2D6 expressing cells) as well as the, level, andbiological activity of expressed gene products (such as 103P2D6 mRNApolynucleotides and polypeptides). Typically, an alteration in thestatus of 103P2D6 comprises a change in the location of 103P2D6 and/or103P2D6 expressing cells and/or an increase in 103P2D6 mRNA and/orprotein expression.

103P2D6 status in a sample can be analyzed by a number of means wellknown in the art, including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, Western blot analysis, and tissue arrayanalysis. Typical protocols for evaluating the status of the 103P2D6gene and gene products are found, for example in Ausubul et al. eds.,1995, Current Protocols In Molecular Biology, Units 2 (NorthernBlotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCRAnalysis). Thus, the status of 103P2D6 in a biological sample isevaluated by various methods utilized by skilled artisans including, butnot limited to genomic Southern analysis (to examine, for exampleperturbations in the 103P2D6 gene), Northern analysis and/or PCRanalysis of 103P2D6 mRNA (to examine, for example alterations in thepolynucleotide sequences or expression levels of 103P2D6 mRNAs), and,Western and/or immunohistochemical analysis (to examine, for examplealterations in polypeptide sequences, alterations in polypeptidelocalization within a sample, alterations in expression levels of103P2D6 proteins and/or associations of 103P2D6 proteins withpolypeptide binding partners). Detectable 103P2D6 polynucleotidesinclude, for example, a 103P2D6 gene or fragment thereof, 103P2D6 mRNA,alternative splice variants, 103P2D6 mRNAs, and recombinant DNA or RNAmolecules containing a 103P2D6 polynucleotide.

The expression profile of 103P2D6 makes it a diagnostic marker for localand/or metastasized disease, and provides information on the growth oroncogenic potential of a biological sample. In particular, the status of103P2D6 provides information useful for predicting susceptibility toparticular disease stages, progression, and/or tumor aggressiveness. Theinvention provides methods and assays for determining 103P2D6 status anddiagnosing cancers that express 103P2D6, such as cancers of the tissueslisted in Table I. For example, because 103P2D6 mRNA is so highlyexpressed in prostate and other cancers relative to normal prostatetissue, assays that evaluate the levels of 103P2D6 mRNA transcripts orproteins in a biological sample can be used to diagnose a diseaseassociated with 103P2D6 dysregulation, and can provide prognosticinformation useful in defining appropriate therapeutic options.

The expression status of 103P2D6 provides information including thepresence, stage and location of dysplastic, precancerous and cancerouscells, predicting susceptibility to various stages of disease, and/orfor gauging tumor aggressiveness. Moreover, the expression profile makesit useful as an imaging reagent for metastasized disease. Consequently,an aspect of the invention is directed to the various molecularprognostic and diagnostic methods for examining the status of 103P2D6 inbiological samples such as those from individuals suffering from, orsuspected of suffering from a pathology characterized by dysregulatedcellular growth, such as cancer.

As described above, the status of 103P2D6 in a biological sample can beexamined by a number of well-known procedures in the art. For example,the status of 103P2D6 in a biological sample taken from a specificlocation in the body can be examined by evaluating the sample for thepresence or absence of 103P2D6 expressing cells (e.g. those that express103P2D6 mRNAs or proteins). This examination can provide evidence ofdysregulated cellular growth, for example, when 103P2D6-expressing cellsare found in a biological sample that does not normally contain suchcells (such as a lymph node), because such alterations in the status of103P2D6 in a biological sample are often associated with dysregulatedcellular growth. Specifically, one indicator of dysregulated cellulargrowth is the metastases of cancer cells from an organ of origin (suchas the prostate) to a different area of the body (such as a lymph node).In this context, evidence of dysregulated cellular growth is importantfor example because occult lymph node metastases can be detected in asubstantial proportion of patients with prostate cancer, and suchmetastases are associated with known predictors of disease progression(see, e.g., Murphy et al., Prostate 42(4): 315–317 (2000);Su et al.,Semin. Surg. Oncol. 18(1): 17–28 (2000) and Freeman et al., J Urol 1995August;154(2 Pt 1):474–8).

In one aspect, the invention provides methods for monitoring 103P2D6gene products by determining the status of 103P2D6 gene productsexpressed by cells from an individual suspected of having a diseaseassociated with dysregulated cell growth (such as hyperplasia or cancer)and then comparing the status so determined to the status of 103P2D6gene products in a corresponding normal sample. The presence of aberrant103P2D6 gene products in the test sample relative to the normal sampleprovides an indication of the presence of dysregulated cell growthwithin the cells of the individual.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in 103P2D6 mRNA or protein expression in a testcell or tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of 103P2D6 mRNA can, for example, beevaluated in tissue samples including but not limited to those listed inTable I. The presence of significant 103P2D6 expression in any of thesetissues is useful to indicate the emergence, presence and/or severity ofa cancer, since the corresponding normal tissues do not express 103P2D6mRNA or express it at lower levels.

In a related embodiment, 103P2D6 status is determined at the proteinlevel rather than at the nucleic acid level. For example, such a methodcomprises determining the level of 103P2D6 protein expressed by cells ina test tissue sample and comparing the level so determined to the levelof 103P2D6 expressed in a corresponding normal sample. In oneembodiment, the presence of 103P2D6 protein is evaluated, for example,using immunohistochemical methods. 103P2D6 antibodies or bindingpartners capable of detecting 103P2D6 protein expression are used in avariety of assay formats well known in the art for this purpose.

In a further embodiment, one can evaluate the status 103P2D6 nucleotideand amino acid sequences in a biological sample in order to identifyperturbations in the structure of these molecules. These perturbationscan include insertions, deletions, substitutions and the like. Suchevaluations are useful because perturbations in the nucleotide and aminoacid sequences are observed in a large number of proteins associatedwith a growth dysregulated phenotype (see, e.g., Marrogi et al., 1999,J. Cutan. Pathol. 26(8):369–378). For example, a mutation in thesequence of 103P2D6 may be indicative of the presence or promotion of atumor. Such assays therefore have diagnostic and predictive value wherea mutation in 103P2D6 indicates a potential loss of function or increasein tumor growth.

A wide variety of assays for observing perturbations in nucleotide andamino acid sequences are well known in the art. For example, the sizeand structure of nucleic acid or amino acid sequences of 103P2D6 geneproducts are observed by the Northern, Southern, Western, PCR and DNAsequencing protocols discussed herein. In addition, other methods forobserving perturbations in nucleotide and amino acid sequences such assingle strand conformation polymorphism analysis are well known in theart (see, e.g., U.S. Pat. No. 5,382,510 issued 7 Sep. 1999, and U.S.Pat. No. 5,952,170 issued 17 Jan. 1995).

Additionally, one can examine the methylation status of the 103P2D6 genein a biological sample. Aberrant demethylation and/or hypermethylationof CpG islands in gene 5′ regulatory regions frequently occurs inimmortalized and transformed cells, and can result in altered expressionof various genes. For example, promoter hypermethylation of the pi-classglutathione S-transferase (a protein expressed in normal prostate butnot expressed in >90% of prostate carcinomas) appears to permanentlysilence transcription of this gene and is the most frequently detectedgenomic alteration in prostate carcinomas (De Marzo et al., Am. J.Pathol. 155(6): 1985–1992 (1999)). In addition, this alteration ispresent in at least 70% of cases of high-grade prostatic intraepithelialneoplasia (PIN) (Brooks et al, Cancer Epidemiol. Biomarkers Prev., 1998,7:531–536). In another example, expression of the LAGE-I tumor specificgene (which is not expressed in normal prostate but is expressed in25–50% of prostate cancers) is induced by deoxy-azacytidine inlymphoblastoid cells, suggesting that tumoral expression is due todemethylation (Lethe et al., Int. J. Cancer 76(6): 903–908 (1998)). Avariety of assays for examining methylation status of a gene are wellknown in the art. For example, one can utilize, in Southernhybridization approaches, methylation-sensitive restriction enzymeswhich cannot cleave sequences that contain methylated CpG sites toassess the methylation status of CpG islands. In addition, MSP(methylation specific PCR) can rapidly profile the methylation status ofall the CpG sites present in a CpG island of a given gene. Thisprocedure involves initial modification of DNA by sodium bisulfite(which will convert all unmethylated cytosines to uracil) followed byamplification using primers specific for methylated versus unmethylatedDNA. Protocols involving methylation interference can also be found forexample in Current Protocols In Molecular Biology, Unit 12, Frederick M.Ausubul et al. eds., 1995.

Gene amplification is an additional method for assessing the status of103P2D6. Gene amplification is measured in a sample directly, forexample, by conventional Southern blotting or Northern blotting toquantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad.Sci. USA, 77:5201–5205), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies are employed thatrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turnare labeled and the assay carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for thepresence of cancer cells using for example, Northern, dot blot or RT-PCRanalysis to detect 103P2D6 expression. The presence of RT-PCRamplifiable 103P2D6 mRNA provides an indication of the presence ofcancer. RT-PCR assays are well known in the art. RT-PCR detection assaysfor tumor cells in peripheral blood are currently being evaluated foruse in the diagnosis and management of a number of human solid tumors.In the prostate cancer field, these include RT-PCR assays for thedetection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol.Res. 25:373–384; Ghossein et al., 1995, J. Clin Oncol. 13:1195–2000;Heston et al., 1995, Clin. Chem. 41:1687–1688).

A further aspect of the invention is an assessment of the susceptibilitythat an individual has for developing cancer. In one embodiment, amethod for predicting susceptibility to cancer comprises detecting103P2D6 mRNA or 103P2D6 protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of 103P2D6 mRNAexpression correlates to the degree of susceptibility. In a specificembodiment, the presence of 103P2D6 in prostate or other tissue isexamined, with the presence of 103P2D6 in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor). Similarly, one can evaluate theintegrity 103P2D6 nucleotide and amino acid sequences in a biologicalsample, in order to identify perturbations in the structure of thesemolecules such as insertions, deletions, substitutions and the like. Thepresence of one or more perturbations in 103P2D6 gene products in thesample is an indication of cancer susceptibility (or the emergence orexistence of a tumor).

The invention also comprises methods for gauging tumor aggressiveness.In one embodiment, a method for gauging aggressiveness of a tumorcomprises determining the level of 103P2D6 mRNA or 103P2D6 proteinexpressed by tumor cells, comparing the level so determined to the levelof 103P2D6 mRNA or 103P2D6 protein expressed in a corresponding normaltissue taken from the same individual or a normal tissue referencesample, wherein the degree of 103P2D6 mRNA or 103P2D6 protein expressionin the tumor sample relative to the normal sample indicates the degreeof aggressiveness. In a specific embodiment, aggressiveness of a tumoris evaluated by determining the extent to which 103P2D6 is expressed inthe tumor cells, with higher expression levels indicating moreaggressive tumors. Another embodiment is the evaluation of the integrityof 103P2D6 nucleotide and amino acid sequences in a biological sample,in order to identify perturbations in the structure of these moleculessuch as insertions, deletions, substitutions and the like. The presenceof one or more perturbations indicates more aggressive tumors.

Another embodiment of the invention is directed to methods for observingthe progression of a malignancy in an individual over time. In oneembodiment, methods for observing the progression of a malignancy in anindividual over time comprise determining the level of 103P2D6 mRNA or103P2D6 protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of 103P2D6 mRNA or 103P2D6 proteinexpressed in an equivalent tissue sample taken from the same individualat a different time, wherein the degree of 103P2D6 mRNA or 103P2D6protein expression in the tumor sample over time provides information onthe progression of the cancer. In a specific embodiment, the progressionof a cancer is evaluated by determining 103P2D6 expression in the tumorcells over time, where increased expression over time indicates aprogression of the cancer. Also, one can evaluate the integrity 103P2D6nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like, where the presence ofone or more perturbations indicates a progression of the cancer.

The above diagnostic approaches can be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention is directed to methods forobserving a coincidence between the expression of 103P2D6 gene and103P2D6 gene products (or perturbations in 103P2D6 gene and 103P2D6 geneproducts) and a factor that is associated with malignancy, as a meansfor diagnosing and prognosticating the status of a tissue sample. A widevariety of factors associated with malignancy can be utilized, such asthe expression of genes associated with malignancy (e.g. PSA, PSCA andPSM expression for prostate cancer etc.) as well as gross cytologicalobservations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol.6(2):74–88; Eptsein, 1995, Hum. Pathol. 26(2):223–9; Thorson et al.,1998, Mod. Pathol. 11(6):543–51; Baisden et al., 1999, Am. J. Surg.Pathol. 23(8):918–24). Methods for observing a coincidence between theexpression of 103P2D6 gene and 103P2D6 gene products (or perturbationsin 103P2D6 gene and 103P2D6 gene products) and another factor that isassociated with malignancy are useful, for example, because the presenceof a set of specific factors that coincide with disease providesinformation crucial for diagnosing and prognosticating the status of atissue sample.

In one embodiment, methods for observing a coincidence between theexpression of 103P2D6 gene and 103P2D6 gene products (or perturbationsin 103P2D6 gene and 103P2D6 gene products) and another factor associatedwith malignancy entails detecting the overexpression of 103P2D6 mRNA orprotein in a tissue sample, detecting the overexpression of PSA mRNA orprotein in a tissue sample (or PSCA or PSM expression), and observing acoincidence of 103P2D6 mRNA or protein and PSA mRNA or proteinoverexpression (or PSCA or PSM expression). In a specific embodiment,the expression of 103P2D6 and PSA mRNA in prostate tissue is examined,where the coincidence of 103P2D6 and PSA mRNA overexpression in thesample indicates the existence of prostate cancer, prostate cancersusceptibility or the emergence or status of a prostate tumor.

Methods for detecting and quantifying the expression of 103P2D6 mRNA orprotein are described herein, and standard nucleic acid and proteindetection and quantification technologies are well known in the art.Standard methods for the detection and quantification of 103P2D6 mRNAinclude in situ hybridization using labeled 103P2D6 riboprobes, Northernblot and related techniques using 103P2D6 polynucleotide probes, RT-PCRanalysis using primers specific for 103P2D6, and other amplificationtype detection methods, such as, for example, branched DNA, SISBA, TMAand the like. In a specific embodiment, semi-quantitative RT-PCR is usedto detect and quantify 103P2D6 mRNA expression. Any number of primerscapable of amplifying 103P2D6 can be used for this purpose, includingbut not limited to the various primer sets specifically describedherein. In a specific embodiment, polyclonal or monoclonal antibodiesspecifically reactive with the wild-type 103P2D6 protein can be used inan immunohistochemical assay of biopsied tissue.

IX.) Identifying Molecules that Interact with 103P2D6

The 103P2D6 protein and nucleic acid sequences disclosed herein allow askilled artisan to identify proteins, small molecules and other agentsthat interact with 103P2D6, as well as pathways activated by 103P2D6 viaany one of a variety of art accepted protocols. For example, one canutilize one of the so-called interaction trap systems (also referred toas the “two-hybrid assay”). In such systems, molecules interact andreconstitute a transcription factor which directs expression of areporter gene, whereupon the expression of the reporter gene is assayed.Other systems identify protein-protein interactions in vivo throughreconstitution of a eukaryotic transcriptional activator, see, e.g.,U.S. Pat. No. 5,955,280 issued 21 Sep. 1999, U.S. Pat. No. 5,925,523issued 20 Jul. 1999, U.S. Pat. No. 5,846,722 issued 8 Dec. 1998 and U.S.Pat. No. 6,004,746 issued 21 Dec. 1999.

Alternatively one can screen peptide libraries to identify moleculesthat interact with 103P2D6 protein sequences. In such methods, peptidesthat bind to a molecule such as 103P2D6 are identified by screeninglibraries that encode a random or controlled collection of amino acids.Peptides encoded by the libraries are expressed as fusion proteins ofbacteriophage coat proteins, the bacteriophage particles are thenscreened against the protein of interest.

Accordingly, peptides having a wide variety of uses, such astherapeutic, prognostic or diagnostic reagents, are thus identifiedwithout any prior information on the structure of the expected ligand orreceptor molecule. Typical peptide libraries and screening methods thatcan be used to identify molecules that interact with 103P2D6 proteinsequences are disclosed for example in U.S. Pat. No. 5,723,286 issued 3Mar. 1998 and U.S. Pat No. 5,733,731 issued 31 Mar. 1998.

Alternatively, cell lines that express 103P2D6 are used to identifyprotein-protein interactions mediated by 103P2D6. Such interactions canbe examined using immunoprecipitation techniques (see, e.g., Hamilton BJ, et al. Biochem. Biophys. Res. Commun. 1999, 261:646–51). 103P2D6protein can be immunoprecipitated from 103P2D6-expressing cell linesusing anti-103P2D6 antibodies. Alternatively, antibodies against His-tagcan be used in a cell line engineered to express 103P2D6 (vectorsmentioned above). The immunoprecipitated complex can be examined forprotein association by procedures such as Western blotting,³⁵S-methionine labeling of proteins, protein microsequencing, silverstaining and two-dimensional gel electrophoresis.

Small molecules and ligands that interact with 103P2D6 can be identifiedthrough related embodiments of such screening assays. For example, smallmolecules can be identified that interfere with protein function,including molecules that interfere with 103P2D6's ability to mediatephosphorylation and de-phosphorylation, second messenger signaling ortumorigenesis. Similarly, ligands that regulate 103P2D6 function can beidentified based on their ability to bind 103P2D6 and activate areporter construct. Typical methods are discussed for example in U.S.Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods for forminghybrid ligands in which at least one ligand is a small molecule. In anillustrative embodiment, cells engineered to express a fusion protein of103P2D6 and a DNA-binding protein are used to co-express a fusionprotein of a hybrid ligand/small molecule and a cDNA librarytranscriptional activator protein. The cells further contain a reportergene, the expression of which is conditioned on the proximity of thefirst and second fusion proteins to each other, an event that occursonly if the hybrid ligand binds to target sites on both hybrid proteins.Those cells that express the reporter gene are selected and the unknownsmall molecule or the unknown ligand is identified. This method providesa means of identifying both activators and inhibitors of 103P2D6.

An embodiment of this invention comprises a method of screening for amolecule that interacts with an 103P2D6 amino acid sequence shown inFIG. 2 and FIG. 3, comprising the steps of contacting a population ofmolecules with the 103P2D6 amino acid sequence, allowing the populationof molecules and the 103P2D6 amino acid sequence to interact underconditions that facilitate an interaction, determining the presence of amolecule that interacts with the 103P2D6 amino acid sequence, and thenseparating molecules that do not interact with the 103P2D6 amino acidsequence from molecules that do. In a specific embodiment, the methodfurther comprises purifying a molecule that interacts with the 103P2D6amino acid sequence. The identified molecule can be used to modulate afunction performed by 103P2D6. In a preferred embodiment, the 103P2D6amino acid sequence is contacted with a library of peptides.

X.) Therapeutic Methods and Compositions

The identification of 103P2D6 as a protein that is normally expressed ina restricted set of tissues, but which is also expressed in prostate andother cancers, opens a number of therapeutic approaches to the treatmentof such cancers. As discussed herein, it is possible that 103P2D6functions as a transcription factor involved in activatingtumor-promoting genes or repressing genes that block tumorigenesis.

Accordingly, therapeutic approaches that inhibit the activity of the103P2D6 protein are useful for patients suffering a cancer thatexpresses 103P2D6. These therapeutic approaches generally fall into twoclasses. One class comprises various methods for inhibiting the bindingor association of the 103P2D6 protein with its binding partner or withothers proteins. Another class comprises a variety of methods forinhibiting the transcription of the 103P2D6 gene or translation of103P2D6 mRNA.

X.A.) 103P2D6 as a Target for Antibody-Based Therapy

103P2D6 is an attractive target for antibody-based therapeuticstrategies. A number of antibody strategies are known in the art fortargeting both extracellular and intracellular molecules (see, e.g.,complement and ADCC mediated killing as well as the use of intrabodies).Because 103P2D6 is expressed by cancer cells of various lineages and notby corresponding normal cells, systemic administration of103P2D6-immunoreactive compositions are prepared that exhibit excellentsensitivity without toxic, non-specific and/or non-target effects causedby binding of the immunoreactive composition to non-target organs andtissues. Antibodies specifically reactive with domains of 103P2D6 areuseful to treat 103P2D6-expressing cancers systemically, either asconjugates with a toxin or therapeutic agent, or as naked antibodiescapable of inhibiting cell proliferation or function.

103P2D6 antibodies can be introduced into a patient such that theantibody binds to 103P2D6 and modulates a function , such as aninteraction with a binding partner, and consequently mediatesdestruction of the tumor cells and/or inhibits the growth of the tumorcells. Mechanisms by which such antibodies exert a therapeutic effectcan include complement-mediated cytolysis, antibody-dependent cellularcytotoxicity, modulation of the physiological function of 103P2D6,inhibition of ligand binding or signal transduction pathways, modulationof tumor cell differentiation, alteration of tumor angiogenesis factorprofiles, and/or apoptosis.

Those skilled in the art understand that antibodies can be used tospecifically target and bind immunogenic molecules such as animmunogenic region of the 103P2D6 sequence shown in FIG. 2. In addition,skilled-artisans understand that it is routine to conjugate antibodiesto cytotoxic agents. When cytotoxic and/or therapeutic agents aredelivered directly to cells, such as by conjugating them to antibodiesspecific for a molecule expressed by that cell (e.g. 103P2D6), thecytotoxic agent will exert its known biological effect (i.e.cytotoxicity) on those cells.

A wide variety of compositions and methods for using antibody-cytotoxicagent conjugates to kill cells are known in the art. In the context ofcancers, typical methods entail administering to an animal having atumor a biologically effective amount of a conjugate comprising aselected cytotoxic and/or therapeutic agent linked to a targeting agent(e.g. an anti-103P2D6 antibody) that binds to a marker (e.g. 103P2D6)expressed, accessible to binding or localized on the cell surfaces. Atypical embodiment is a method of delivering a cytotoxic and/ortherapeutic agent to a cell expressing 103P2D6, comprising conjugatingthe cytotoxic agent to an antibody that immunospecifically binds to a103P2D6 epitope, and, exposing the cell to the antibody-agent conjugate.Another illustrative embodiment is a method of treating an individualsuspected of suffering from metastasized cancer, comprising a step ofadministering parenterally to said individual a pharmaceuticalcomposition comprising a therapeutically effective amount of an antibodyconjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-103P2D6 antibodies can be done inaccordance with various approaches that have been successfully employedin the treatment of other types of cancer, including but not limited tocolon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133–138),multiple myeloma (Ozaki et al., 1997, Blood 90:3179–3186, Tsunenari etal., 1997, Blood 90:2437–2444), gastric cancer (Kasprzyk et al., 1992,Cancer Res. 52:2771–2776), B-cell lymphoma (Funakoshi et al., 1996, J.Immunother. Emphasis Tumor Immunol. 19:93–101), leukemia (Zhong et al.,1996,-Leuk. Res. 20:581–589), colorectal cancer (Moun et al., 1994,Cancer Res. 54:6160–6166; Velders et al., 1995, Cancer Res.55:4398–4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117–127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin, such as the conjugation of ¹³¹I to anti-CD20antibodies (e.g., Rituxan™, IDEC Pharmaceuticals Corp.), while othersinvolve co-administration of antibodies and other therapeutic agents,such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). Totreat prostate cancer, for example, 103P2D6 antibodies can beadministered in conjunction with radiation, chemotherapy or hormoneablation.

Although 103P2D6 antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of 103P2D6expression, preferably using immunohistochemical assessments of tumortissue, quantitative 103P2D6 imaging, or other techniques that reliablyindicate the presence and degree of 103P2D6 expression.Immunohistochemical analysis of tumor biopsies or surgical specimens ispreferred for this purpose. Methods for immunohistochemical analysis oftumor tissues are well known in the art.

Anti-103P2D6 monoclonal antibodies that treat prostate and other cancersinclude those that initiate a potent immune response against the tumoror those that are directly cytotoxic. In this regard, anti-103P2D6monoclonal antibodies (mAbs) can elicit tumor cell lysis by eithercomplement-mediated or antibody-dependent cell cytotoxicity (ADCC)mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fe receptorsites on complement proteins. In addition, anti-103P2D6 mAbs that exerta direct biological effect on tumor growth are useful to treat cancersthat express 103P2D6. Mechanisms by which directly cytotoxic mAbs actinclude: inhibition of cell growth, modulation of cellulardifferentiation, modulation of tumor angiogenesis factor profiles, andthe induction of apoptosis. The mechanism(s) by which a particularanti-103P2D6 mAb exerts an anti-tumor effect is evaluated using anynumber of in vitro assays that evaluate cell death such as ADCC, ADMMC,complement-mediated cell lysis, and so forth, as is generally known inthe art.

In some patients, the use of murine or other non-human monoclonalantibodies, or human/mouse chimeric mAbs can induce moderate to strongimmune responses against the non-human antibody. This can result inclearance of the antibody from circulation and reduced efficacy. In themost severe cases, such an immune response can lead to the extensiveformation of immune complexes which, potentially, can cause renalfailure. Accordingly, preferred monoclonal antibodies used in thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 103P2D6antigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-103P2D6 mAbs as well as combinations, or cocktails, ofdifferent mAbs. Such mAb cocktails can have certain advantages inasmuchas they contain mAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic mAbs with mAbs thatrely on immune effector functionality. Such mAbs in combination canexhibit synergistic therapeutic effects. In addition, anti-103P2D6 mAbscan be administered concomitantly with other therapeutic modalities,including but not limited to various chemotherapeutic agents,androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery orradiation. The anti-103P2D6 mAbs are administered in their “naked” orunconjugated form, or can have a therapeutic agent(s) conjugated tothem.

Anti-103P2D6 antibody formulations are administered via any routecapable of delivering the antibodies to a tumor cell. Routes ofadministration include, but are not limited to, intravenous,intraperitoneal, intramuscular, intratumor, intradermal, and the like.Treatment generally involves repeated administration of the anti-103P2D6antibody preparation, via an acceptable route of administration such asintravenous injection (IV), typically at a dose in the range of about0.1 to about 10 mg/kg body weight. In general, doses in the range of10–500 mg mAb per week are effective and well tolerated.

Based on clinical experience with the Herceptin mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kgIV of the anti-103P2D6 mAb preparation represents an acceptable dosingregimen. Preferably, the initial loading dose is administered as a 90minute or longer infusion. The periodic maintenance dose is administeredas a 30 minute or longer infusion, provided the initial dose was welltolerated. As appreciated by those of skill in the art, various factorscan influence the ideal dose regimen in a particular case. Such factorsinclude, for example, the binding affinity and half life of the Ab ormAbs used, the degree of 103P2D6 expression in the patient, the extentof circulating shed 103P2D6 antigen, the desired steady-state antibodyconcentration level, frequency of treatment, and the influence ofchemotherapeutic or other agents used in combination with the treatmentmethod of the invention, as well as the health status of a particularpatient.

Optionally, patients should be evaluated for the levels of 103P2D6 in agiven sample (e.g. the levels of circulating 103P2D6 antigen and/or103P2D6 expressing cells) in order to assist in the determination of themost effective dosing regimen, etc. Such evaluations are also used formonitoring purposes throughout therapy, and are useful to gaugetherapeutic success in combination with the evaluation of otherparameters (such as serum PSA levels in prostate cancer therapy).

X.B.) Anti-Cancer Vaccines

The invention further provides cancer vaccines comprising a103P2D6-related protein or 103P2D6-related nucleic acid. In view of theexpression of 103P2D6, cancer vaccines prevent and/or treat103P2D6-expressing cancers without creating non-specific effects onnon-target tissues. The use of a tumor antigen in a vaccine thatgenerates humoral and/or cell-mediated immune responses as anti-cancertherapy is well known in the art and has been employed in prostatecancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995,Int. J. Cancer 63:231–237; Fong et al., 1997, J. Immunol.159:3113–3117).

Genetic immunization methods can be employed to generate prophylactic ortherapeutic humoral and cellular immune responses directed againstcancer cells expressing 103P2D6. Constructs comprising DNA encoding a103P2D6-related protein/immunogen and appropriate regulatory sequencescan be injected directly into muscle or skin of an individual, such thatthe cells of the muscle or skin take-up the construct and express theencoded 103P2D6 protein/immunogen. Alternatively, a vaccine comprises a103P2D6-related protein. Expression of the 103P2D6-related proteinimmunogen results in the generation of prophylactic or therapeutichumoral and cellular immunity against cells that bear 103P2D6 protein.Various prophylactic and therapeutic genetic immunization techniquesknown in the art can be used (for review, see information and referencespublished at Internet address www.genweb.com).

Such methods can be readily practiced by employing a 103P2D6-relatedprotein, or an 103P2D6-encoding nucleic acid molecule and recombinantvectors capable of expressing and presenting the 103P2D6 immunogen(which typically comprises a number of antibody or T cell epitopes).Skilled artisans understand that a wide variety of vaccine systems fordelivery of immunoreactive epitopes are known in the art (see, e.g.,Heryln et al., Ann Med 1999 Feburary;31(1):66–78; Maruyama et al.,Cancer Immunol Immunother 2000 June;49(3):123–32) Briefly, such methodsof generating an immune response (e.g. humoral and/or cell-mediated) ina mammal, comprise the steps of: exposing the mammal's immune system toan immunoreactive epitope (e.g. an epitope present in the 103P2D6protein shown in SEQ ID NO: 2 or analog or homolog thereof) so that themammal generates an immune response that is specific for that epitope(e.g. generates antibodies that specifically recognize that epitope). Ina preferred method, the 103P2D6 immunogen contains a biological motif.

CTL epitopes can be determined using specific algorithms to identifypeptides within 103P2D6 protein that are capable of optimally binding tospecified HLA alleles (e.g., Table IV (A) and Table IV (B); Epimer™ andEpimatrix™, Brown University(http://www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html);and, BIMAS, (http://bimas.dcrt.nih.gov/). In a preferred embodiment, the103P2D6 immunogen contains one or more amino acid sequences identifiedusing one of the pertinent analytical techniques well known in the art,such as the sequences shown in Tables V–XVIII or a peptide of 8, 9, 10or 11 amino acids specified by an HLA Class I motif (e.g., Table IV (A))and/or a peptide of at least 9 amino acids that comprises an HLA ClassII motif (e.g., Table IV (B)). As is appreciated in the art, the HLAClass I binding grove is essentially closed ended so that peptides ofonly a particular size range can fit into the groove and be bound,generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. Incontrast, the HLA Class II binding groove is essentially open ended;therefore a of about 9 or more amino acids can be bound by an HLA ClassII molecule. Due to the binding groove differences between HLA Class Iand II, HLA Class I motifs are length specific, i.e., position two of aClass I motif is the second amino acid in an amino to carboxyl directionof the peptide. The amino acid positions in a Class II motif arerelative only to each other, not the overall peptide, i.e., additionalamino acids can be attached to the amino and/or carboxyl termini of amotif-bearing sequence. HLA Class II epitopes are often 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 24, or 25 amino acids long,but can be longer than 25 amino acids.

A wide variety of methods for generating an immune response in a mammalare known in the art (for example as the first step in the generation ofhybridomas). Methods of generating an immune response in a mammalcomprise exposing the mammal's immune system to an immunogenic epitopeon a protein (e.g. the 103P2D6 protein) so that an immune response isgenerated. A typical embodiment consists of a method for generating animmune response to 103P2D6 in a host, by contacting the host with asufficient amount of at least one 103P2D6 B cell or cytotoxic T-cellepitope or analog thereof; and at least one periodic interval thereafterre-contacting the host with the 103P2D6 B cell or cytotoxic T-cellepitope or analog thereof. A specific embodiment consists of a method ofgenerating an immune response against a 103P2D6-related protein or aman-made multiepitopic peptide comprising: administering 103P2D6immunogen (e.g. the 103P2D6 protein or a peptide fragment thereof, an103P2D6 fusion protein or analog etc.) in a vaccine preparation to ahuman or another mammal. Typically, such vaccine preparations furthercontain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or auniversal helper epitope such as a PADRE™ peptide (Epimmune Inc., SanDiego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3);164(3): 1625–1633; Alexander et al., Immunity 1994 1(9): 751–761 andAlexander et al., Immunol. Res. 1998 18(2): 79–92). An alternativemethod comprises generating an immune response in an individual againsta 103P2D6 immunogen by: administering in vivo to muscle or skin of theindividual's body a DNA molecule that comprises a DNA sequence thatencodes an 103P2D6 immunogen, the DNA sequence operatively linked toregulatory sequences which control the expression of the DNA sequence;wherein the DNA molecule is taken up by cells, the DNA sequence isexpressed in the cells and an immune response is generated against theimmunogen (see, e.g., U.S. Pat. No. 5,962,428). The DNA can bedissociated from an infectious agent. Optionally a genetic vaccinefacilitator such as anionic lipids; saponins; lectins; estrogeniccompounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea isalso administered.

Thus, viral gene delivery systems are used to deliver a 103P2D6-relatednucleic acid molecule. Various viral gene delivery systems that can beused in the practice of the invention include, but are not limited to,vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus,adeno-associated virus, lentivirus, and sindbus virus (Restifo, 1996,Curr. Opin. Inmunol. 8:658–663). Non-viral delivery systems can also beemployed by introducing naked DNA encoding a 103P2D6-related proteininto the patient (e.g., intramuscularly or intradermally) to induce ananti-tumor response. In one embodiment, the full-length human 103P2D6cDNA is employed. In another embodiment, 103P2D6 nucleic acid moleculesencoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopesare employed.

Various ex vivo strategies can also be employed to generate an immuneresponse. One approach involves the use of antigen presenting cells(APCs) such as dendritic cells to present 103P2D6 antigen to a patient'simmune system. Dendritic cells express MHC class I and II molecules, B7co-stimulator, and IL-12, and are thus highly specialized antigenpresenting cells. In prostate cancer, autologous dendritic cells pulsedwith peptides of the prostate-specific membrane antigen (PSMA) are beingused in a Phase I clinical trial to stimulate prostate cancer patients'immune systems (Tjoa et aL, 1996, Prostate 28:65–69; Murphy et al.,1996, Prostate 29:371–380). Thus, dendritic cells can be used to present103P2D6 peptides to T cells in the context of MHC class I or IImolecules. In one embodiment, autologous dendritic cells are pulsed with103P2D6 peptides capable of binding to MHC class I and/or class IImolecules. In another embodiment, dendritic cells are pulsed with thecomplete 103P2D6 protein. Yet another embodiment involves engineeringthe overexpression of the 103P2D6 gene in dendritic cells using variousimplementing vectors known in the art, such as adenovirus (Arthur etal., 1997, Cancer Gene Ther. 4:17–25), retrovirus (Henderson et al.,1996, Cancer Res. 56:3763–3770), lentivirus, adeno-associated virus, DNAtransfection (Ribas et al., 1997, Cancer Res. 57:2865–2869), ortumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med.186:1177–1182). Cells that express 103P2D6 can also be engineered toexpress immune modulators, such as GM-CSF, and used as immunizingagents.

Anti-idiotypic anti-103P2D6 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga 103P2D6-related protein. In particular, the generation ofanti-idiotypic antibodies is well known in the art; this methodology canreadily be adapted to generate anti-idiotypic anti-103P2D6 antibodiesthat mimic an epitope on a 103P2D6-related protein (see, for example,Wagner et al., 1997, Hybridoma 16: 3340; Foon et al., 1995, J. Clin.Invest. 96:334–342; Herlyn et al., 1996, Cancer Immunol. Immunother.43:65–76). Such an anti-idiotypic antibody can be used in cancer vaccinestrategies.

XI.) Inhibition of 103P2D6 Protein Function

The invention includes various methods and compositions for inhibitingthe binding of 103P2D6 to its binding partner or its association withother protein(s) as well as methods for inhibiting 103P2D6 function.

XI.A.) Inhibition of 103P2D6 with Intracellular Antibodies

In one approach, a recombinant vector that encodes single chainantibodies that specifically bind to 103P2D6 are introduced into 103P2D6expressing cells via gene transfer technologies. Accordingly, theencoded single chain anti-103P2D6 antibody is expressed intracellularly,binds to 103P2D6 protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, arespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatment isfocused. This technology has been successfully applied in the art (forreview, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodieshave been shown to virtually eliminate the expression of otherwiseabundant cell surface receptors (see, e.g., Richardson et al., 1995,Proc. Natl. Acad. Sci. USA 92: 3137–3141; Beerli et al., 1994, J. Biol.Chem. 289: 23931–23936; Deshane et al., 1994, Gene Ther. 1: 332–337).

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies areexpressed as a single chain variable region fragment joined to the lightchain constant region. Well-known intracellular trafficking signals areengineered into recombinant polynucleotide vectors encoding such singlechain antibodies in order to precisely target the intrabody to thedesired intracellular compartment. For example, intrabodies targeted tothe endoplasmic reticulum (ER) are engineered to incorporate a leaderpeptide and, optionally, a C-terminal ER retention signal, such as theKDEL amino acid motif Intrabodies intended to exert activity in thenucleus are engineered to include a nuclear localization signal. Lipidmoieties are joined to intrabodies in order to tether the intrabody tothe cytosolic side of the plasma membrane. Intrabodies can also betargeted to exert function in the cytosol. For example, cytosolicintrabodies are used to sequester factors within the cytosol, therebypreventing them from being transported to their natural cellulardestination.

In one embodiment, intrabodies are used to capture 103P2D6 in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals are engineered into such 103P2D6 intrabodies in orderto achieve the desired targeting. Such 103P2D6 intrabodies are designedto bind specifically to a particular 103P2D6 domain. In anotherembodiment, cytosolic intrabodies that specifically bind to the 103P2D6protein are used to prevent 103P2D6 from gaining access to the nucleus,thereby preventing it from exerting any biological activity within thenucleus (e.g., preventing 103P2D6 from forming transcription complexeswith other factors).

In order to specifically direct the expression of such intrabodies toparticular cells, the transcription of the intrabody is placed under theregulatory control of an appropriate tumor-specific promoter and/orenhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer can beutilized (See, for example, U.S. Pat. No. 5,919,652 issued Jul. 6,1999).

XI.B.) Inhibition of 103P2D6 with Recombinant Proteins

In another approach, recombinant molecules bind to 103P2D6 and therebyinhibit 103P2D6 function . For example, these recombinant moleculesprevent or inhibit 103P2D6 from accessing/binding to its bindingpartner(s) or associating with other protein(s). Such recombinantmolecules can, for example, contain the reactive part(s) of a 103P2D6specific antibody molecule. In a particular embodiment, the 103P2D6binding domain of a 103P2D6 binding partner is engineered into a dimericfusion protein, whereby the fusion protein comprises two 103P2D6 ligandbinding domains linked to the Fc portion of a human IgG, such as humanIgGI. Such IgG portion can contain, for example, the C_(H)2 and C_(H)3domains and the hinge region, but not the C_(H)1 domain. Such dimericfusion proteins are administered in soluble form to patients sufferingfrom a cancer associated with the expression of 103P2D6, whereby thedimeric fusion protein specifically binds to 103P2D6 and blocks 103P2D6interaction with a binding partner. Such dimeric fusion proteins arefurther combined into multimeric proteins using known antibody linkingtechnologies.

XI.C.) Inhibition of 103P2D6 Transcription or Translation

The present invention also comprises various methods and compositionsfor inhibiting the transcription of the 103P2D6 gene. Similarly, theinvention also provides methods and compositions for inhibiting thetranslation of 103P2D6 mRNA into protein.

In one approach, a method of inhibiting the transcription of the 103P2D6gene comprises contacting the 103P2D6 gene with a 103P2D6 antisensepolynucleotide. In another approach, a method of inhibiting 103P2D6 mRNAtranslation comprises contacting the 103P2D6 mRNA with an antisensepolynucleotide. In another approach, a 103P2D6 specific ribozyme is usedto cleave the 103P2D6 message, thereby inhibiting translation. Suchantisense and ribozyme based methods can also be directed to theregulatory regions of the 103P2D6 gene, such as the 103P2D6 promoterand/or enhancer elements. Similarly, proteins capable of inhibiting a103P2D6 gene transcription factor are used to inhibit 103P2D6 mRNAtranscription. The various polynucleotides and compositions useful inthe aforementioned methods have been described above. The use ofantisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

Other factors that inhibit the transcription of 103P2D6 by interferingwith 103P2D6 transcriptional activation are also useful to treat cancersexpressing 103P2D6. Similarly, factors that interfere with 103P2D6processing are useful to treat cancers that express 103P2D6. Cancertreatment methods utilizing such factors are also within the scope ofthe invention.

XI.D.) General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used to delivertherapeutic polynucleotide molecules to tumor cells synthesizing 103P2D6(i.e., antisense, ribozyme, polynucleotides encoding intrabodies andother 103P2D6 inhibitory molecules). A number of gene therapy approachesare known in the art. Recombinant vectors encoding 103P2D6 antisensepolynucleotides, ribozymes, factors capable of interfering with 103P2D6transcription, and so forth, can be delivered to target tumor cellsusing such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a widevariety of surgical, chemotherapy or radiation therapy regimens. Thetherapeutic approaches of the invention can enable the use of reduceddosages of chemotherapy (or other therapies) and/or less frequentadministration, an advantage for all patients and particularly for thosethat do not tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, can beevaluated using various in vitro and in vivo assay systems. In vitroassays that evaluate therapeutic activity include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 103P2D6 to a bindingpartner, etc.

In vivo, the effect of a 103P2D6 therapeutic composition can beevaluated in a suitable animal model. For example, xenogenic prostatecancer models can be used, wherein human prostate cancer explants orpassaged xenograft tissues are introduced into immune compromisedanimals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine3: 402–408). For example, PCT Patent Application WO98/16628, Sawyers etal., published Apr. 23, 1998, describes various xenograft models ofhuman prostate cancer capable of recapitulating the development ofprimary tumors, micrometastasis, and the formation of osteoblasticmetastases characteristic of late stage disease. Efficacy can bepredicted using assays that measure inhibition of tumor formation, tumorregression or metastasis, and the like. In vivo assays that evaluate thepromotion of apoptosis are useful in evaluating therapeuticcompositions. In one embodiment, xenografts from tumor bearing micetreated with the therapeutic composition can be examined for thepresence of apoptotic foci and compared to untreated controlxenograft-bearing mice. The extent to which apoptotic foci are found inthe tumors of the treated mice provides an indication of the therapeuticefficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed.,1980).

Therapeutic formulations can be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations can be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancer,and will generally depend on a number of other factors appreciated inthe art.

XII.) Kits

For use in the diagnostic and therapeutic applications described herein,kits are also within the scope of the invention. Such kits can comprisea carrier, package or container that is compartmentalized to receive oneor more containers such as vials, tubes, and the like, each of thecontainer(s) comprising one of the separate elements to be used in themethod. For example, the container(s) can comprise a probe that is orcan be detectably labeled. Such probe can be an antibody orpolynucleotide specific for a 103P2D6-related protein or a 103P2D6 geneor message, respectively. Where the method utilizes nucleic acidhybridization to detect the target nucleic acid, the kit can also havecontainers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradioisotope label. The kit can include all or part of the amino acidsequence of FIG. 2 or analogs thereof, or a nucleic acid molecule thatencodes such amino acid sequences.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse.

A label can be present on the container to indicate that the compositionis used for a specific therapy or non-therapeutic application, and canalso indicate directions for either in vivo or in vitro use, such asthose described above. Directions and or other information can also beincluded on an insert which is included with the kit.

p103P2D6-B (clone B) has been deposited under the requirements of theBudapest Treaty on May 19, 2000 with the American Type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209 USA,and has been identified as ATCC Accession No. PTA-1895. p103P2D6-2(clone 2) has been deposited under the requirements of the BudapestTreaty on Jan. 6, 2000 with the American Type Culture Collection (ATCC),10801 University Blvd., Manassas, Va. 20110-2209 USA, and has beenidentified as ATCC Accession No. PTA-1155.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which are intendedto limit the scope of the invention.

Example 1 SSH-generated Isolation of a cDNA Fragment of the 103P2D6 Gene

Characterization of 103P2D6 by Suppression Subtractive Hybridization(SSH)

As discussed in detail below, experiments with the LAPC-4 AD xenograftin male SCID mice have resulted in the identification of genes that areinvolved in the progression of androgen dependent (AD) prostate cancerto androgen independent (AI) cancer. Briefly, mice that harbored LAPC-4AD xenografts were castrated when the tumors reached a size of 1 cm indiameter. The tumors regressed in size and temporarily stopped producingthe androgen dependent protein PSA. Seven to fourteen dayspost-castration, PSA levels were detectable again in the blood of themice. Eventually such tumors develop an AI phenotype and start growingagain in the castrated males. Tumors were harvested at different timepoints after castration to identify genes that are turned on or offduring the transition to androgen independence.

Suppression subtractive hybridization (SSH) (Diatchenko et al., 1996,PNAS 93:6025) was then used to identify novel genes, such as those thatare overexpressed in prostate cancer, by comparing cDNAs from variousandrogen dependent and androgen independent LAPC xenografts. Thisstrategy resulted in the identification of novel genes exhibitingprostate cancer specific expression. One of these genes, designated103P2D6, was identified from a subtraction where cDNA derived from anLAPC-4 AD tumor grown in an intact male was subtracted from cDNA derivedfrom an LAPC4 AD tumor, 21 days post-castration. The SSH DNA sequence ofabout 342 bp (FIG. 1) is novel and exhibits no homology to sequences inthe public databases.

103P2D6, encodes a transmembrane protein that exhibits tumor-specificexpression. Expression analysis of 103P2D6 indicates that it isexclusively expressed in cancer of the prostate and other cancertissues. 103P2D6 is expressed in fetal heart, kidney and lung, but notin normal adult tissues. The expression of 103P2D6 in cancer providesevidence that this protein has a functional role in tumor progressionand/or initiation. It is possible that 103P2D6 function s as a receptorinvolved in activating or modulating proliferation signals involved intumorigenesis and regulation of cell growth.

As is further described herein, the 103P2D6 gene and protein have beencharacterized using a number of analytical approaches. For example,analyses of nucleotide coding and amino acid sequences were conducted inorder to identify potentially related molecules, as well as recognizablestructural domains, topological features, and other elements within the103P2D6 mRNA and protein structures. Northern blot analyses of 103P2D6mRNA expression were conducted in order to establish the range of normaland cancerous tissues expressing 103P2D6 message.

Materials and Methods

LAPC Xenografts and Human Tissues:

LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) andgenerated as described (Klein et al, 1997, Nature Med. 3: 402–408; Craftet al., 1999, Cancer Res. 59: 5030–5036). Androgen dependent andindependent LAPC-4 xenografts (LAPC-4 AD and AI, respectively) andLAPC-9 xenografts (LAPC-9 AD and AI, respectively) were grown in intactmale SCID mice or in castrated males, respectively, and were passaged assmall tissue chunks in recipient males. LAPC4 AI xenografts were derivedfrom LAPC4 AD tumors and LAPC-9 AI xenografts were derived from LAPC-9AD tumors. To generate the AI xenografts, male mice bearing LAPC ADtumors were castrated and maintained for 2–3 months. After the LAPCtumors re-grew, the tumors were harvested and passaged in castratedmales or in female SCID mice.

Cell Lines:

Human cell lines (e.g., HeLa) were obtained from the ATCC and weremaintained in DMEM with 5% fetal calf serum.

RNA Isolation:

Tumor tissue and cell lines were homogenized in Trizol reagent (LifeTechnologies, Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cells toisolate total RNA. Poly A RNA was purified from total RNA using Qiagen'sOligotex mRNA Mini and Midi kits. Total and mRNA were quantified byspectrophotometric analysis (O.D. 260/280 nm) and analyzed by gelelectrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA synthesis primer): 5′TTTTGATCAAGCTT₃₀3′ (SEQ ID NO: 7)Adaptor 1: 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ ID NO: 8)                              3′GGCCCGTCCTAG5′ (SEQ ID NO: 9) Adaptor 2:5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO: 10)                           3′CGGCTCCTAG5′ (SEQ ID NO: 11) PCR primer 1:5′CTAATACGACTCACTATAGGGC3′ (SEQ ID NO: 12) Nested primer (NP)1:5′TCGAGCGGCCGCCCGGGCAGGA3′ (SEQ ID NO: 13) Nested primer (NP)2:5′AGCGTGGTCGCGGCCGAGGA3′ (SEQ ID NO: 14)Suppression Subtractive Hybridization:

Suppression subtractive hybridization (SSH) was used to identify cDNAscorresponding to genes that may be differentially expressed in prostatecancer. The SSH reaction utilized cDNA from two LAPC-4 AD xenografts.Specifically, the 103P2D6 SSH sequence was identified from a subtractionwhere cDNA derived from an LAPC-4 AD tumor grown in an intact male wassubtracted from cDNA derived from an LAPC-4 AD tumor, 21 dayspost-castration. The LAPC-4 AD xenograft tumor, 21 days post-castration,was used as the source of the “tester” cDNA, while the cDNA from theLAPC-4 AD tumor grown in an intact male was used as the source of the“driver” cDNA.

Double stranded cDNAs corresponding to tester and driver cDNAs weresynthesized from 2 μg of poly(A)⁺ RNA isolated from the relevantxenograft tissue, as described above, using CLONTECH's PCR-Select cDNASubtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- andsecond-strand synthesis were carried out as described in the Kit's usermanual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1).The resulting cDNA was digested with Dpn II for 3 hrs. at 37° C.Digested cDNA was extracted with phenol/chloroform (1:1) and ethanolprecipitated.

Driver cDNA was generated by combining in a 1:1 ratio DpnII digestedcDNA from the relevant xenograft source (see above) with a mix ofdigested cDNAs derived from human benign prostatic hyperplasia (BPH),the human cell lines HeLa, 293, A431, Colo205, and mouse liver.

Tester cDNA was generated by diluting 1 μl of DpnII digested cDNA fromthe relevant xenograft source (see above) (400 ng) in 5 μl of water. Thediluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 andAdaptor 2 (10 μM), in separate ligation reactions, in a total volume of10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH).Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C.for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10–12 cycles of 94° C. for 10sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR productswere analyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dbest, and NCI-CGAP databases.

Results

Two SSH experiments described in the Materials and Methods, supra, ledto the isolation of numerous candidate gene fragment clones (SSHclones). All candidate clones were sequenced and subjected to homologyanalysis against all sequences in the major public gene and ESTdatabases in order to provide information on the identity of thecorresponding gene and to help guide the decision to analyze aparticular gene for differential expression. In general, gene fragmentsthat had no homology to any known sequence in any of the searcheddatabases, and thus considered to represent novel genes, as well as genefragments showing homology to previously sequenced expressed sequencetags (ESTs), were subjected to differential expression analysis byRT-PCR and/or northern analysis.

One of the SHH clones comprising about 342 bp, showed no homology to anyknown gene or to any sequences in the public databases, and wasdesignated 103P2D6. Northern expression analysis of first strand cDNAsfrom 16 normal tissues showed a highly prostate tumor-specificexpression pattern in adult tissues (FIG. 4).

Example 2 Full Length Cloning of 103P2D6 and Homology Comparison toKnown Sequences

A partial 103P2D6 cDNA clone (clone 2) of 1687 base pairs (bp) wascloned from an LAPC-4 AD cDNA library (Lambda ZAP Express, Stratagene).A full-length 103P2D6 cDNA clone (FIG. 2) (clone B) of 4728 base pairs(b.p.) was cloned from a human fetal brain library (Pangene Inc.). ThecDNA encodes a putative open reading frame (ORF) of 563 amino acids. Itscalculated molecular weight (MW) is 63.4 kDa and its pI is 8.15. At theprotein level, 103P2D6 shows 24.9% identity and 32.8% homology, takingaccount of any gaps, to an Envelope protein (Q9UNM3) isolated from ahuman endogenous retroviral protein HERV-H (Virology 1999, 258:441). The103P2D6 nucleic acid sequence overlaps with some ESTs derived fromkidney, fetus, brain and placenta.

The full-length 103P2D6 cDNA (clone B) was deposited on May 19, 2000,with the American Type Culture Collection (ATCC; Manassas, Va.) asplasmid p103P2D6-B, and has been assigned Accession No. PTA-1895. Thepartial 103P2D6 cDNA (clone 2) was deposited on Jan. 6, 2000, with theAmerican Type Culture Collection (ATCC; Manassas, Va.) as plasmidp103P2D6-2, and has been assigned Accession No. PTA-1155.

Example 3 103P2D6 Gene & Protein Expression Analysis

Northern Expression Analysis:

103P2D6 mRNA expression in normal human tissues was analyzed by northernblotting of multiple tissue blots (Clontech; Palo Alto, Calif.),comprising a total of 16 different normal human tissues, using labeled103P2D6 SSH fragment (Example 1) as a probe. RNA samples werequantitatively normalized with a , β-actin probe. Northern blot analysisusing an 103P2D6 SSH fragment probe performed on 16 normal tissuesfailed to show expression in any normal tissues, including brain, ovary,heart, lung, liver, kidney, pancreas, small intestine, placenta,leukocytes, testis, prostate, colon, skeletal muscle, thymus and spleen(FIG. 5).

To analyze 103P2D6 expression in cancer tissues, northern blotting wasperformed on RNA derived from the LAPC xenografts, and several prostateand non-prostate cancer cell lines. The results show high expressionlevels of a 3 kb transcript in LAPC-4 AD, LAPC-4 AI, LAPC-9 AD, andLAPC-9 AI (FIG. 5, FIG. 6). More detailed analysis of the LAPC-4xenografts shows that 103P2D6 is expressed at equal levels whether thexenografts are grown subcutaneously (LAPC-4 sc) or within the tibia ofmice (LAPC4 AD it) (FIG. 7). Expression was also detected in a xenograftthat was grown within human bone explants in SCID mice (the LAPC-4 AD₂).This indicates that bone growth of these prostate cancer tissues doesnot diminish their expression.

Expression of 103P2D6 was detected in several cancer cell lines derivedfrom prostate (LNCaP, DU145, LAPC-4 CL), bladder (HT1197, 5637),pancreas (BxPC-3, HPAC, CAPAN-1), colon (SK-CO-1, CACO-2), bone (HOS,U2-OS, RD-ES), lung (CALU-1), breast (MCF-7), testis (NCCIT, TERA-1),cervix (A431), and ovary (OV-1063, PA-1, SW626) (FIG. 6). These resultssuggest that the 103P2D6 is generally up-regulated in cancer cells andcancer tissues, especially in prostate, kidney and bladder cancer, andserves as a suitable target for cancer therapy.

Northern blot analysis of 103P2D6 showed expression only in cancertissues and cell lines, but not in normal adult tissues. Furthermore,the 103P2D6 gene was cloned from a fetal brain library, suggesting thatit is a fetal gene that is activated during tumorigenesis. Toinvestigate this hypothesis, 9 week old fetal RNA derived from 6 organsand from the whole embryo were analyzed by Northern blotting using a103P2D6 SSH probe. The results (FIG. 8) show that 103P2D6 is expressedin fetal heart, kidney and lung at the 9 week stage. This indicates that103P2D6 is indeed a fetal gene that is turned on in cancer and serves arole in cancer biology.

Expression of 103P2D6 was assayed in a panel of human cancers (T) andtheir respective matched normal tissues (N) on RNA dot blots (FIG. 13).103P2D6 expression was seen in cancers of the kidney, breast, prostate,uterus, ovary, cervix, colon, stomach and rectum 103P2D6 was also foundto be highly expressed in the two human cancer cell lines, the CML lineK562 and the colorectal carcinoma SW480. The expression detected innormal adjacent tissues (isolated from diseased tissues) but not innormal tissues, isolated from healthy donors, indicates that thesetissues are not fully normal and that 103P2D6 may be expressed in earlystage tumors and that it has utility as a diagnostic marker.

FIG. 14 shows Northern data where RNA was isolated from prostate tumors(T) and their adjacent normal tissues (N) obtained from the followingprostate cancer patients (Pt); patient 1, Gleason score 4+5; patient 2,Gleason score 3+4; and, patient 3, Gleason score 4+3. Northern analysiswas performed using 10 μg of total RNA for each sample. Expression of103P2D6 was seen in all three tumor samples tested and their respectivenormal tissues.

Data from a Northern analysis where RNA was isolated from kidney tumors(T) and their adjacent normal tissues (N) obtained from kidney cancerpatients is shown in FIG. 15. The patient specifications are as follows:Patient 1—Papillary Type, Stage I, Grade 2/4; Patient 2—Invasivepapillary carcinoma, Grade 2/4; Patient 3—Clear cell type Grade 1/3,focally 2/3; Patient 4—Clear cell type, stage III, Grade 2/4; Patient5—Clear cell type, stage III, Grade 3/4; Patient 6—Clear cell type,stage III, Grade 3/4; Patient 7—Clear cell type, Grade III. In FIG. 15,CL=Cell lines (from left to right): 769-P, A498, SW839; NK=Normalkidney; N=Normal adjacent tissue; T=Tumor. The Northern analysis wasperformed using 10 μg of total RNA for each sample. Elevated expressionof 103P2D6 was observed in kidney tumors and normal adjacent tissuesisolated from kidney cancer patients as compared to normal kidney.

FIG. 16 shows the results of Northern analysis where RNA was isolatedfrom bladder cancers and adjacent normal tissue obtained from thebladder cancer patients. The Northern analysis was performed using 10 μgof total RNA for each sample. Expression of 103P2D6 was seen in bladdertumor but not in normal adjacent tissue.

RT-PCR Expression Analysis:

First strand cDNAs can be generated from 1 μg of mRNA with oligo (dT)12–18 priming using the Gibco-BRL Superscript Preamplification system.The manufacturer's protocol was used, which included an incubation for50 min at 42° C. with reverse transcriptase, followed by RNAse Htreatment at 37° C. for 20 min. After completing the reaction, thevolume can be increased to 200 μl with water prior to normalization.First strand cDNAs from 16 different normal human tissues can beobtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′-ATATCGCCGCGCTCGTCGTCGACAA-3′ (SEQ IDNO: XX) and 5′-AGCCACACGCAGCTCATTGTAGAAGG-3′ (SEQ ID NO: XX) to amplifyβ-actin. First strand cDNA (5 μl) were amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech,10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1× Klentaq DNApolymerase (Clontech). Five μl of the PCR reaction can be removed at 18,20, and 22 cycles and used for agarose gel electrophoresis. PCR wasperformed using an MJ Research thermal cycler under the followingconditions: Initial denaturation can be at 94° C. for 15 sec, followedby a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C.for 5 sec. A final extension at 72° C. was carried out for 2 min. Afteragarose gel electrophoresis, the band intensities of the 283 b.p.β-actin bands from multiple tissues were compared by visual inspection.Dilution factors for the first strand cDNAs were calculated to result inequal β-actin band intensities in all tissues after 22 cycles of PCR.Three rounds of normalization can be required to achieve equal bandintensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 103P2D6 gene, 5 μl of normalizedfirst strand cDNA were analyzed by PCR using 25, 30, and 35 cycles ofamplification. Semi quantitative expression analysis can be achieved bycomparing the PCR products at cycle numbers that give light bandintensities.

In a typical RT-PCR expression analysis shown in FIG. 9, RT-PCRexpression analysis was performed on first strand cDNAs generated usingpools of tissues from multiple samples. The cDNAs were subsequentlynormalized using beta-actin PCR. The highest expression was observed incolon cancer pool, xenograft pool, and a lung cancer patient. Lowerlevels of expression were also observed normal prostate, prostatecancer, bladder cancer, and kidney cancer tissue pools.

In an additional analysis, RT-PCR was used to analyze expression of103P2D6 in normal tissues and in patient-derived cancers. First strandcDNAs were generated from 1 μg of mRNA with oligo (dT)12–18 primingusing the Gibco-BRL Superscript Preamplification system. Themanufacturers protocol was used and included an incubation for 50 min at42° C. with reverse transcriptase followed by RNAse H treatment at 37°C. for 20 min. After completing the reaction, the volume was increasedto 200 μl with water prior to normalization. First strand cDNAs wereprepared from normal prostate, normal kidney and HeLa cancer cells, aswell as a prostate tumor pool, a kidney tumor pool and a bladder tumorpool. The tumor pools were prepared from patient-derived tumor tissue.Normalization was performed by PCR using primers to actin and GAPDH.Semi-quantitative PCR was performed using primers to 103P2D6. The103P2D6 primers used for RT-PCR were: CTTGGGAGGTCCTAGTGCTAAGTG (SEQ. IDNO: ) and CAATGAAGGGACTAACAACCCATC (SEQ. ID NO: XX). The results areshown in FIG. 9, and confirm that 103P2D6 is expressed in canceroustissues, particularly in prostate and bladder cancer tissues derivedfrom patients.

Protein Expression Analysis:

Expression of 103P2D6 protein was analyzed in pancreatic, colon, andprostate cancer cell lines using both western blot and flow cytometricanalysis. As shown in FIG. 11A–B, cell lysates (˜25 μg) from theindicated cell lines were separated by SDS-PAGE and subjected to Westernblot analysis using an anti-103P2D6 pAb (see Example 4, below).Indicated with an arrow is a strong anti-103P2D6 pAb immunoreactive bandof approximately 60 kD present in the pancreatic cancer cell lines HPACand Bx PC-3, the colon cancer cell line CaCo-2, and a less intense bandin LAPC9 prostate cancer cells, indicative of endogenous 103P2D6 proteinexpression. Also indicated with an arrow is the 85 kD immunoreactiveband present in 293T cells transfected with V5-His tagged 103P2D6 cDNA.

Bx PC-3 pancreatic cancer cells were stained with anti-103P2D6 pAb (10μml) or control rabbit IgG Ab and subjected to flow cytometric analysisfollowing incubation with anti-rabbit IgG-FITC conjugated secondary Ab.Bx PC-3 cells stained with the anti-103P2D6 pAb exhibited a fluorescenceshift compared to the cells stained with control rabbit IgG, indicatingcell surface expression of 103P2D6. The results are shown in FIG. 11C.

Example 4 Generation of 103P2D6 Polyclonal Antibodies

Generation of Polyclonal Antibodies (pAbs)

To generate polyclonal antibodies (pAb) to 103P2D6, a peptide wassynthesized corresponding to amino acids 163–176 (DVTNESRNDDDDTS) of the103P2D6 protein sequence. The peptide was coupled to Keyhole limpethemacyanin (KLH) and used to immunize a rabbit as follows. The rabbitwas initially immunized with 200 μg of peptide-KLH mixed in completeFreund's adjuvant. The rabbit was then injected every two to three weekswith 200 μg of peptide-KLH in incomplete Freund's adjuvant. Bleeds weretaken approximately 7–10 days following each immunization. The titer ofthe serum was at least 1:64,000 as determined by ELISA to the peptide.

Affinity purified 103P2D6 polyclonal antibodies were prepared by passageof crude serum from the immunized rabbit over an affinity matrixcomprised of 103P2D6 peptide covalently coupled to Affigel 10 (BioRad).After extensive washing of the matrix with PBS, antibodies specific to103P2D6 peptide were eluted with low pH glycine buffer (0.1M, pH 2.5).Western blotting using the affinity purified pAb revealed the appearanceof an anti-103P2D6 immunoreactive band of approximately 85 kD in 293Tcells transiently transfected with the 103P2D6 cDNA in the pCDNA 3.1V5-His vector, but not with the control empty vector (FIG. 12A). ThispAb also detected cell surface expression of 103P2D6 protein intransfected 293T cells as determined by flow cytometry (FIG. 12B).

PAbs are also prepared by immunization of mice, rabbits, goats, andsheep with recombinant bacterial fusion proteins encoding full length orvarious regions of the 103P2D6 sequence. The recombinant bacterialproteins include glutathione-S-transferase (GST), maltose bindingprotein (MBP), and HIS tagged fusion proteins of 103P2D6 that arepurified from induced bacteria using the appropriate affinity matrix.Mammalian expressed secreted Tag5 or FC-fusion proteins encoding theextracellular domain are also used as immunogens for pAb production.

Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Forexample, 103P2D6, recombinant bacterial fusion proteins or peptidesencoding various regions of the 103P2D6 sequence are used to immunizeNew Zealand White rabbits. Typically a peptide can be designed from acoding region of 103P2D6. The peptide can be conjugated to keyholelimpet hemocyanin (KLH) and used to immunize a rabbit. Alternatively theimmunizing agent may include all or portions of the 103P2D6 protein,analogs or fusion proteins thereof. For example, the 103P2D6 amino acidsequence can be fused to any one of a variety of fusion protein partnersthat are well known in the art, such as maltose binding protein, LacZ,thioredoxin or an immunoglobulin constant region (see e.g. CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubulet al. eds., 1995; Linsley, P. S., Brady, W., Umes, M., Grosmaire, L.,Damle, N., and Ledbetter, L.(1991) J. Exp. Med. 174, 561–566). Otherrecombinant bacterial proteins include glutathione-S-transferase (GST),and HIS tagged fusion proteins of 103P2D6 that are purified from inducedbacteria using the appropriate affinity matrix.

It may be useful to conjugate the immunizing agent to a protein known tobe immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate).

In a typical protocol, rabbits are initially immunized subcutaneouslywith about 200 μg of fusion protein or peptide conjugated to KLH mixedin complete Freund's adjuvant. Rabbits are then injected subcutaneouslyevery two weeks with 200 μg of immunogen in incomplete Freund'sadjuvant. Test bleeds are taken approximately 7–10 days following eachimmunization and used to monitor the titer of the antiserum by ELISA.

To test serum, such as rabbit serum, for reactivity with 103P2D6proteins, the full-length 103P2D6 cDNA can be cloned into an expressionvector such as one that provides a 6His tag at the carboxyl-terminus(pCDNA 3.1 myc-his, Invitrogen). After transfection of the constructsinto 293T cells, cell lysates can be probed with anti-His antibody(Santa Cruz Biotechnologies, Santa Cruz, Calif.) and the anti-103P2D6serum using Western blotting. Alternatively specificity of the antiserumis tested by Western blot and immunoprecipitation analyses using lysatesof cells that express 103P2D6. Serum from rabbits immunized with GST orMBP fusion proteins is first semi-purified by removal of anti-GST oranti-MBP antibodies by passage over GST and MBP protein columnsrespectively. Sera from His-tagged protein and peptide immunized rabbitsas well as depleted GST and MBP protein sera are purified by passageover an affinity column composed of the respective immunogen covalentlycoupled to Affigel matrix (BioRad).

Example 5 Production of Recombinant 103P2D6 in Bacterial and MammalianSystems

Bacterial Constructs

pGEX Constructs

To express 103P2D6 in bacterial cells, portions of 103P2D6 were fused tothe glutathione S-transferase (GST) gene by cloning into pGEX-6P-1(Amersham Pharmacia Biotech, NJ). The constructs were made in order togenerate recombinant 103P2D6 protein sequences with GST fused at theN-terminus and a six histidine epitope at the C-terminus. The sixhistidine epitope tag is generated by adding the histidine codons to thecloning primer at the 3′ end of the open reading frame (ORF). APreScission™ recognition site permits cleavage of the GST tag from103P2D6-related protein. The ampicillin resistance gene and pBR322origin permits selection and maintenance of the plasmid in E. coli. Forexample, cDNA encoding the following fragments of 103P2D6 protein werecloned into pGEX-6P-1: amino acids 24 to 487; amino acids 39 to 176;amino acids 170 to 360; and amino acids 1 to 536, or any 8, 9, 10, 11,12,13, 14 or 15 contiguous amino acids from 103P2D6 or an analogthereof.

pMAL Constructs

To express 103P2D6in bacterial cells, all or part of the 103P2D6nucleicacid sequence are fused to the maltose-binding protein (MBP) gene bycloning into pMAL-c2X and pMAL-p2X (New England Biolabs, MA). Theconstructs are made to generate recombinant 103P2D6protein sequenceswith MBP fused at the N-terminus and a six histidine epitope at theC-terminus. The six histidine epitope tag is generated by adding thehistidine codons to the 3′ cloning primer. A Factor Xa recognition sitepermits cleavage of the GST tag from 103P2D6. The pMAL-c2X and pMAL-p2Xvectors are optimized to express the recombinant protein in thecytoplasm or periplasm respectively. Periplasm expression enhancesfolding of proteins with disulfide bonds. For example, cDNA encoding thefollowing fragments of 103P2D6 protein are cloned into pGEX-6P-1: aminoacids 24 to 487; amino acids 39 to 176; amino acids 170 to 360; andamino acids 1 to 536, or any 8, 9, 10, 11, 12, 13, 14 or 15 contiguousamino acids from 103P2D6 or an analog thereof.

pCRII

To generate 103P2D6 sense and anti-sense riboprobes for RNA in situinvestigations, a pCRII construct was generated using cDNA sequenceencoding amino acids 44–181. The pCRII vector has Sp6 and T7 promotersflanking the insert to drive the production of 108P5H8 RNA riboprobeswhich will be used in RNA in situ hybridization experiments.

Mammalian Constructs

To express recombinant 103P2D6, the full or partial length 103P2D6cDNAcan be cloned into any one of a variety of expression vectors known inthe art. The constructs can be transfected into any one of a widevariety of mammalian cells such as 293T cells. Transfected 293T celllysates can be probed with the anti-103P2D6polyclonal serum, describedin Example 4 above, in a Western blot.

The 103P2D6genes can also be subcloned into the retroviral expressionvector pSRαMSVtkneo and used to establish 103P2D6-expressing cell linesas follows: The 103P2D6 coding sequence (from translation initiation ATGand Kozak translation start consensus sequence to the terminationcodons) is amplified by PCR using ds cDNA template from 103P2D6 cDNA.The PCR product is subcloned into pSRαMSVtkneo vector and transformedinto DH5α competent cells. Colonies are picked to screen for clones withunique internal restriction sites on the cDNA. The positive clone isconfirmed by sequencing of the cDNA insert. The retroviral vectors canthereafter be used for infection and generation of various cell linesusing, for example, NIH 3T3, TsuPr1, 293 or rat-1 cells.

Additional illustrative mammalian and bacterial systems are discussedbelow.

pcDNA4/HisMax-TOPO Constructs

To express 103P2D6 in mammalian cells, the 103P2D6 ORF is cloned intopcDNA4/HisMax-TOPO Version A (cat# K864-20, Invitrogen, Carlsbad,Calif.). Protein expression is driven from the cytomegalovirus (CMV)promoter and the SP163 translational enhancer. The recombinant proteinhas Xpress™ and six histidine epitopes fused to the N-terminus to aid indetection and purification of the recombinant protein. ThepcDNA4/HisMax-TOPO vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheZeocin resistance gene allows for selection of mammalian cellsexpressing the protein and the ampicillin resistance gene and ColE1origin permits selection and maintenance of the plasmid in E. coli.

pcDNA3.1/MycHis Constructs

To express 103P2D6 in mammalian cells, the ORF with consensus Kozaktranslation initiation site is cloned into pcDNA3.1/MycHis_Version A(Invitrogen, Carlsbad, Calif.). Protein expression is driven from thecytomegalovirus (CMV) promoter. The recombinant protein has the mycepitope and six histidines fused to the C-terminus to aid in detectionand purification of the recombinant protein. The pcDNA3.1/MycHis vectoralso contains the bovine growth hormone (BGH) polyadenylation signal andtranscription termination sequence to enhance mRNA stability, along withthe SV40 origin for episomal replication and simple vector rescue incell lines expressing the large T antigen. The Neomycin resistance genecan be used, as it allows for selection of mammalian cells expressingthe protein and the ampicillin resistance gene and ColE1 origin permitsselection and maintenance of the plasmid in E. coli.

pcDNA 3.1/V5His-TOPO Constructs

To express 103P2D6 in mammalian cells, the cDNA encoding the 103P2D6 ORFand Kozak consensus translation initiation sequence was cloned intopcDNA4/V5His-TOPO (cat# K4800-01, Invitrogen, Carlsbad, Calif.). Proteinexpression is driven from the cytomegalovirus (CMV) promoter. Therecombinant protein has V5™ and six histidine epitopes fused at theC-terminus to aid in detection and purification of the recombinantprotein. The pcDNA4/V5His-TOPO vector also contains the bovine growthhormone (BGH) polyadenylation signal and transcription terminationsequence to enhance mRNA stability along with the SV40 origin forepisomal replication and simple vector rescue in cell lines expressingthe large T antigen. The Zeocin resistance gene allows for selection ofmammalian cells expressing the protein and the ampicillin resistancegene and ColE1 origin permits selection and maintenance of the plasmidin E. coli.

Protein Expression

The mammalian expression vector pcDNA 3.1 V5-His encoding the 103P2D6cDNA was used to transfect 293T human embryonic kidney cells to assess103P2D6 protein expression. Western analysis of cell lysates using ananti-103P2D6 pAb reveals an immunoreactive band of approximately 85 kDseen in 293T cells transiently transfected with the 103P2D6 cDNA but notin cells transfected with the control empty vector (FIG. 12A). Flowcytometric analysis of the same cells stained with the anti-103P2D6 pAbreveals a fluorescent shift of the 103P2D6 transfected cells compared tocontrol cells indicating cell surface expression of the 103P2D6 protein(FIG. 12B). Western analysis of 103P2D6 mRNA positive HPAC and Bx PC-3pancreatic cancer cells, CaCo-2 colon cancer cells, and LAPC9 prostatecancer cells, reveals expression of a 60 kD anti-103P2D6 immunoreactiveband indicating significant endogenous expression of the 103P2D6 protein(Example 3, above; FIG. 11A–B). The increased molecular weight of103P2D6 in transfected 293T cells (85 kD) may be due to the presence ofthe V5-His amino acid tag present in the cDNA or post-translationalprocessing or modification of 103P2D6 when overexpressed in 293T cells.103P2D6 protein is expressed on the cell surface of Bx PC-3 cells asindicated by flow cytometric analysis (Example 3, above; FIG. 11C)

pcDNA3.1CT-GFP-TOPO Construct

To express 103P2D6 in mammalian cells and to allow detection of therecombinant protein using fluorescence, the ORF with consensus Kozaktranslation initiation site is cloned into pcDNA3.1CT-GFP-TOPO(Invitrogen, Calif.). Protein expression is driven from thecytomegalovirus (CMV) promoter. The recombinant protein has the GreenFluorescent Protein (GFP) fused to the C-terminus facilitatingnon-invasive, in vivo detection and cell biology studies. ThepcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheNeomycin resistance gene allows for selection of mammalian cells thatexpress the protein, and the ampicillin resistance gene and ColE1 originpermits selection and maintenance of the plasmid in E. coli. Anadditional construct with a N-terminal GFP fusion is made inpcDNA3.1NT-GFP-TOPO spanning the entire length of the 103P2D6protein.

pAPtag Constructs

The cDNA encoding 103P2D6 amino acids 24–487 is cloned into pAPtag-5(GenHunter Corp. Nashville, Tenn.). This construct generates an alkalinephosphatase fusion at the C-terminus of the 103P2D6protein while fusingthe IgGK signal sequence to N-terminus. The resulting recombinant103P2D6 protein is optimized for secretion into the media of transfectedmammalian cells and can be used to identify proteins such as ligands orreceptors that interact with the 103P2D6protein. Protein expression isdriven from the CMV promoter and the recombinant protein also containsmyc and six histidines fused to the C-terminus of alkaline phosphataseto aid in detection and purification of the recombinant protein. TheZeosin resistance gene allows for selection of mammalian cellsexpressing the protein and the ampicillin resistance gene permitsselection of the plasmid in E. coli.

ptag5 Constructs

The cDNA encoding for 103P2D6 amino acids 24–487, 24–174, 24–233, and234–487, was cloned into pTag-5. This vector is similar to pAPtag butwithout the alkaline phosphatase fusion. This construct generates animmunoglobulin G1 Fc fusion at the C-terminus of the 103P2D6proteinwhile fusing the IgGK signal sequence to the N-terminus. The resultingrecombinant 103P2D6 protein is optimized for secretion into the media oftransfected mammalian cells, and can be used to identify proteins suchas ligands or receptors that interact with the 103P2D6protein. Proteinexpression is driven from the CMV promoter and the recombinant proteinalso contains myc and six histidines fused to the C-terminus to aid indetection and purification of the recombinant protein. The Zeocinresistance gene allows for selection of mammalian cells expressing theprotein, and the ampicillin resistance gene permits selection of theplasmid in E. coli.

psecFc Constructs

The cDNA encoding for 103P2D6 amino acids 24–487, 24–233, and 234–487,was cloned into psecFc. The psecFc vector was assembled by cloningimmunoglobulin G1 Fc (hinge, CH2, CH3 regions) into pSecTag2(Invitrogen, California). This construct generates an immunoglobulin G1Fc fusion at the C-terminus of the 103P2D6protein, while fusing the IgGKsignal sequence to N-terminus. The resulting recombinant 103P2D6 proteinis optimized for secretion into the media of transfected mammaliancells, and can be used to identify proteins such as ligands or receptorsthat interact with the 103P2D6 protein. Protein expression is drivenfrom the CMV promoter. The Zeocin resistance gene allows for selectionof mammalian cells that express the protein, and the ampicillinresistance gene permits selection of the plasmid in E. coli.

pSRαConstructs

To generate mammalian cell lines that express 103P2D6 constitutively,the ORF was cloned into pSRα constructs. Amphotropic and ecotropicretroviruses are generated by transfection of pSRα constructs into the293T-10A1 packaging line or co-transfection of pSRα and a helper plasmid(φ˜) in the 293 cells, respectively. The retrovirus can be used toinfect a variety of mammalian cell lines, resulting in the integrationof the cloned gene, 103P2D6, into the host cell-lines. Proteinexpression is driven from a long terminal repeat (LTR). The Neomycinresistance gene allows for selection of mammalian cells that express theprotein, and the ampicillin resistance gene and ColE1 origin permitselection and maintenance of the plasmid in E. coli.

An additional pSRα construct was made that fused the FLAG tag to theC-terminus to allow detection using anti-FLAG antibodies. The FLAGsequence 5′ gat tac aag gat gac gac gat aag 3′ (SEQ ID NO: XX) was addedto cloning primer at the 3′ end of the ORF.

Additional pSRα constructs are made to produce both N-terminal andC-terminal GFP and myc/6 HIS fusion proteins of the full-length103P2D6protein.

Example 6 Production of Recombinant 103P2D6in a Baculovirus System

To generate a recombinant 103P2D6 protein in a baculovirus expressionsystem, cDNA sequence encoding the 103P2D6 protein is cloned into thebaculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides aHis-tag at the N-terminus Specifically, pBlueBac--103P2D6 isco-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9(Spodoptera frugiperda) insect cells to generate recombinant baculovirus(see Invitrogen instruction manual for details). Baculovirus is thencollected from cell supernatant and purified by plaque assay.

Recombinant 103P2D6protein is then generated by infection of HighFiveinsect cells (Invitrogen) with the purified baculovirus. Recombinant103P2D6 protein can be detected using anti-103P2D6 antibody. 103P2D6protein can be purified and used in various cell-based assays or asimmunogen to generate polyclonal and monoclonal antibodies specific for103P2D6.

Example 7 Chromosomal Mapping of the 103P2D6 Gene

The chromosomal localization of 103P2D6 was determined using theGeneBridge4 Human/Hamster radiation hybrid (RH) panel (Walter et al.,1994, Nat. Genetics 7:22) (Research Genetics, Huntsville Ala.).

The following PCR primers were used to localize 103P2D6:

103P2D6.1 cttgggaggtcctagtgctaagtg 103P2D6.2 caatgaagggactaacaacccatc

The resulting mapping vector for the 93 radiation hybrid panel DNAs was:010001110101101001101111110011200100100001001110011111001000101101100100110110110110101000011. This vector and the mapping program athttp://www-genome.wi.mit.edu/cgi -bin/contig/rhmapper.pl placed 103P2D6on chromosome 4p12–p14 (D4S3197-D4S1577).

Example 8 Identification of Potential Signal Transduction Pathways

Based on its surface localization, 103P2D6 can regulate key signalingpathways. Several pathways known to play a role in cancer biology can beregulated by 103P2D6, including phospholipid pathways such as P13K, AKT,etc, as well as mitogenic/survival cascades such as ERK, p38, etc (CellGrowth Differ. 2000,11:279; J Biol Chem. 1999, 274:801; Oncogene. 2000,19:3003.). The role that 103P2D6 plays in the regulation of thesepathways can be investigated using, Western blotting techniques andreporter assays. Cells lacking 103P2D6 and cells expressing 103P2D6 areeither left untreated or stimulated with serum, cytokines, androgen orantibodies. Cell lysates are analyzed using anti-phosphos-specificantibodies (Cell Signaling, Santa Cruz Biotechnology) in order to detectphosphorylation and regulation of ERK, p38, AKT, P13K, PLC and othersignaling molecules. When 103P2D6 plays a role in the regulation ofsignaling pathways, 103P2D6 is used as a target for diagnostic,preventative and therapeutic purposes.

To determine whether 103P2D6 directly or indirectly activates knownsignal transduction pathways in cells, luciferase (luc) basedtranscriptional reporter assays are carried out in cells that express103P2D6. These transcriptional reporters contain consensus binding sitesfor known transcription factors that lie downstream ofwell-characterized signal transduction pathways. Some of the reportersand examples of these associated transcription factors, signaltransduction pathways, and activation stimuli are listed below.

-   1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK: growth/apoptosis/stress-   2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK: growth/differentiation-   3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC: growth/apoptosis/stress-   4. ARE-luc, androgen receptor; steroids/MAPK:    growth/differentiation/apoptosis-   5. p53-luc, p53; SAPK: growth/differentiation/apoptosis-   6. CRE-luc, CREB/ATF2: PKA/p38; growth/apoptosis/stress

When 103P2D6 plays a role in the regulation of growth, apoptosis,stress, or differentiation, 103P2D6 is used as a target for diagnostic,preventative and therapeutic purposes.

103P2D6-mediated effects are assayed in cells showing 103P2D6 mRNAexpression. For example, Luciferase reporter plasmids can be introducedby lipid-mediated transfection (TFX-50, Promega). Luciferase activity,an indicator of relative transcriptional activity, is measured byincubation of cell extracts with luciferin substrate and luminescence ofthe reaction is monitored in a luminometer. This assay allows one todetermine the effect of signaling pathways activation. When 103P2D6plays a role in activation signaling pathways, 103P2D6 is used as atarget for diagnostic, preventative and therapeutic purposes.

Example 9 Generation of 103P2D6 Monoclonal Antibodies (mAbs)

To generate mAbs to 103P2D6, mice are immunized intraperitoneally with10–50 μg of protein immunogen mixed in complete Freund's adjuvant.Protein immunogens include peptides, recombinant 103P2D6 proteins, and,mammalian expressed human IgG FC fusion proteins. Mice are thensubsequently immunized every 24 weeks with 10–50 μg of antigen mixed inFreund's incomplete adjuvant. Alternatively, Ribi adjuvant is used forinitial immunizations. In addition, a DNA-based immunization protocol isused in which a mammalian expression vector used to immunize mice bydirect injection of the plasmid DNA. For example, a pCDNA 3.1 encoding103P2D6 cDNA alone or as an IgG FC fusion is used. This protocol is usedalone or in combination with protein immunogens. Test bleeds are taken7–10 days following immunization to monitor titer and specificity of theimmune response. Once appropriate reactivity and specificity is obtainedas determined by ELISA, Western blotting, and immunoprecipitationanalyses, fusion and hybridoma generation is then carried withestablished procedures well known in the art (Harlow and Lane, 1988).

In an illustrative method for generating 103P2D6 monoclonal antibodies,a glutathione-S-transferase (GST) fusion protein encompassing a 103P2D6protein is synthesized and used as immunogen. Balb C mice are initiallyimmunized intraperitoneally with 200 μg of the GST-103P2D6 fusionprotein mixed in complete Freund's adjuvant. Mice are subsequentlyimmunized every two weeks with 75 μg of GST-103P2D6 protein mixed inFreund's incomplete adjuvant for a total of three immunizations.Reactivity of serum from immunized mice to full-length 103P2D6 proteinis monitored by ELISA using a partially purified preparation ofHIS-tagged 103P2D6 protein expressed from 293T cells (Example 5). Miceshowing the strongest reactivity are rested for three weeks and given afinal injection of fusion protein in PBS and then sacrificed four dayslater. The spleens of the sacrificed mice are then harvested and fusedto SPO/2 myeloma cells using standard procedures (Harlow and Lane,1988). Supernatants from growth wells following HAT selection arescreened by ELISA and Western blot to identify 103P2D6 specificantibody-producing clones.

The binding affinity of a 103P2D6 monoclonal antibody is determinedusing standard technologies. Affinity measurements quantify the strengthof antibody to epitope binding and can be used to help define which103P2D6 monoclonal antibodies are preferred for diagnostic ortherapeutic use. The BIAcore system (Uppsala, Sweden) is a preferredmethod for determining binding affinity. The BIAcore system uses surfaceplasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Mortonand Myszka, 1998, Methods in Enzymology 295: 268) to monitorbiomolecular interactions in real time. BIAcore analysis convenientlygenerates association rate constants, dissociation rate constants,equilibrium dissociation constants, and affinity constants.

Example 10 Involvement of 103P2D6 in Cancer Growth and Progression

The finding that 103P2D6 is differentially expressed in cancer cellssuggests that 103P2D6 participates in the process of tumor formationand/or progression. This is supported by the presence of otherenvelope-like proteins in prostate, colon and endothelial tumor linesbut not normal cells (Pathobiology. 1997, 65:123; Cancer Lett. 1998,124:213). For example, HERV-K, another envelope protein, is expressed interatocarcinoma and tumor cell lines (J Gen Virol. 1996; 77: 2983), andHERV-R is expressed in gonadoblastomas (Virchows Arch. 1999; 434:11).

103P2D6 contributes to the growth of prostate and other cancer cells(See Table I) by several mechanisms. The 103P2D6 protein can function asa cell surface receptor or as a transporter and contribute to tumorgrowth by regulating tumor cell proliferation or responding to tumorcells, endothelial cells or stroma. Alternatively, 103P2D6 contributesto tumor growth by mediating cellular adhesion, transformation ordownstream gene expression. The function s of 103P2D6 can be evaluated,e.g., by using engineered cell lines that express 103P2D6. For example,primary cells such as PrEC, cancer cell lines and NIH 3T3 cellsengineered to stably express 103P2D6 are evaluated for cell growthpotential. When 103P2D6 participates in neoplastic cell growth, 103P2D6is used as a target for diagnostic, preventative and therapeuticpurposes.

Moreover, the role 103P2D6 plays in transformation is evaluated. Primarycells, such as PrEC, and NIH3T3 cells engineered to express 103P2D6 arecompared to 103P2D6-negative cells, to evaluate their ability to formcolonies in soft agar (Song Z. et al. Cancer Res. 2000; 60:6730), wherecolony formation indicates the presence of transformed cells. When103P2D6 mediates transformation, 103P2D6 is used as a target fordiagnostic, preventative and therapeutic purposes.

The expression of 103P2D6 in the various cancers listed in Table Iindicates that this gene has a functional role in tumor progression.103P2D6 function can be assessed in mammalian cells using in vitroapproaches. Mammalian cells infected with the retroviral vectorpSRαtkneo or pSRα-103P2D6 are compared (Muller et al., 1991, MCB 11:1785) using a variety of assays, including cell proliferation in tissueculture and in vitro invasion using a membrane invasion culture system(Welch et al., Int. J. Cancer 43: 449–457). Cell lines expressing103P2D6 are assayed for invasive and migratory properties by measuringpassage of cells through a matrigel coated Transwell™ system (BectonDickinson) (Cancer Res. 1999; 59:6010). Passage of cells through themembrane to the opposite side is monitored using a fluorescent assay(Becton Dickinson Technical Bulletin #428). For example, cells lacking103P2D6 and cells expressing 103P2D6 are loaded with the fluorescentdye, calcein, and plated in the top well of the Transwell insert.Invasion is determined by fluorescence of cells in the lower chamberrelative to the fluorescence of the entire cell population. When 103P2D6is involved with cell invasion, 103P2D6 is used as a target fordiagnostic, preventative and therapeutic purposes.

Envelope proteins have been shown to exhibit chemotactic abilities (LinC L, Sewell A K, Gao fGF, Whelan K T, Phillips R E, Austyn J M. J ExpMed. 2000, 192:587). In view of its similarity to envelope proteins, andto determine whether 103P2D6-expressing cells have such chemoattractantproperties, indicator cells are monitored for passage through theTranswell system toward a gradient of 103P2D6-conditioned media comparedto control media. This assay can also be used to qualify and quantifyspecific neutralization of 103P2D6 effects. For example, theneutralization can be effected by candidate cancer therapeuticcompositions. When 103P2D6 mediates tissue invasion, such as bychemotaction, 103P2D6 is used as a target for diagnostic, preventativeand therapeutic purposes.

The function of 103P2D6 can be evaluated using anti-sense RNA technologycoupled to the various functional assays described herein, e.g. growth,invasion and migration. Anti-sense RNA oligonucleotides can beintroduced into 103P2D6 expressing cells, thereby preventing theexpression of 103P2D6. Control and anti-sense containing cells can beanalyzed for proliferation, invasion, migration, apoptotic andtranscriptional potential. The local as well as systemic effect of theloss of 103P2D6 expression can be evaluated.

Example 11 Protein Association and Cell Adhesion

Envelope proteins have been shown to mediate cell adhesion and syncytiumformation (J. Immunol. 1996, 156:1307; AIDS. 1991, 5:1425). Based on itssimilarity with envelope proteins, 103P2D6 can mediate protein-proteinassociation resulting in multimeric complexes. The association ofproteins into large complexes is critical in several biologicalprocesses, including signal transduction, cell communication,ubiquitination, transcriptional regulation, etc.

Alternatively, 103P2D6 can participate in regulating cell adhesion andcommunication. To determine the degree to which expression of 103P2D6regulates cell adhesion, cells lacking 103P2D6 are compared to cellsexpressing 103P2D6, using techniques known in the art (see, e.g., Haieret al, Br. J. Cancer. 1999, 80:1867; Lehr and Pienta, J. Natl. CancerInst. 1998, 90:118). Briefly, in one embodiment, cells labeled with afluorescent indicator, such as calcein, are incubated on tissue culturewells coated with media alone or with matrix proteins. Adherent cellsare detected by fluorimetric analysis. Confirmation of the role 103P2D6plays in adhesion can be obtained using anti-103P2D6 antibodies. Sincecell adhesion plays a critical role in tumor growth, progression, and,colonization, the inhibition of 103P2D6-mediated interactions serves asa diagnostic, preventative and therapeutic modality.

Example 12 In Vivo Assay for 103P2D6 Tumor Growth Promotion

The effect of the 103P2D6 protein on tumor cell growth can be evaluatedin vivo by gene overexpression in tumor-bearing mice. For example, SCIDmice can be injected SQ on each flank with 1×10⁶ of either PC3, TSUPR1,or DU145 cells containing tkNeo empty vector or 103P2D6. At least twostrategies may be used: (1) Constitutive 103P2D6 expression underregulation of a promoter such as a constitutive promoter obtained fromthe genomes of viruses such as polyoma virus, fowlpox virus (UK2,211,504 published Jul. 5, 1989), adenovirus (such as Adenovirus 2),bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, provided such promoters are compatible with thehost cell systems. (2) Regulated expression under control of aninducible vector system, such as ecdysone, tet, etc., can be usedprovided such promoters are compatible with the host cell systems. Tumorvolume is then monitored at the appearance of palpable tumors and isfollowed over time to determine if 103P2D6-expressing cells grow at afaster rate and whether tumors produced by 103P2D6-expressing cellsdemonstrate characteristics of altered aggressiveness (e.g. enhancedmetastasis, vascularization, reduced responsiveness to chemotherapeuticdrugs). Additionally, mice can be implanted with 1×10⁵ of the same cellsorthotopically to determine if 103P2D6 has an effect on local growth inthe prostate or on the ability of the cells to metastasize, specificallyto lungs, lymph nodes, and bone marrow. Also see saffron et al,“Anti-PSCA mAbs inhibit tumor growth and metastasis formation andprolong the survival of mice bearing human prostate cancer xenografts”PNAS (in press, 2001).

The assay is also useful to determine the 103P2D6 inhibitory effect ofcandidate therapeutic compositions, such as for example, 103P2D6intrabodies, 103P2D6 antisense molecules and ribozymes.

Example 13 Western Analysis of 103P2D6 Expression in SubcellularFractions

The cellular location of 103P2D6 can be assessed using subcellularfractionation techniques widely used in cellular biology (Storrie B, etal. Methods Enzymol. 1990; 182:203–25). Prostate or other cell lines canbe separated into nuclear, cytosolic and membrane fractions. Theexpression of 103P2D6 in the different fractions can be tested usingWestern blotting techniques.

Alternatively, to determine the subcellular localization of 103P2D6,293T cells can be transfected with an expression vector encodingHIS-tagged 103P2D6 (PCDNA 3.1 MYC/HIS, Invitrogen). The transfectedcells can be harvested and subjected to a differential subcellularfractionation protocol as previously described (Pemberton, P. A. et al,1997, J of Histochemistry and Cytochemistry, 45:1697–1706.) Thisprotocol separates the cell into fractions enriched for nuclei, heavymembranes (lysosomes, peroxisomes, and mitochondria), light membranes(plasma membrane and endoplasmic reticulum), and soluble proteins.

Example 14 Localization of 103P2D6

Based on its structure, 103P2D6 is understood to be associated with thecell surface. Surface staining experiments confirm that 103P2D6 cDNA isexpressed at the cell surface (see, e.g., FIGS. 11–12). When located atthe cell membrane, the potential functions of 103P2D6 include (1) asurface receptor that transmits signals to the cell nucleus, or (2) atransporter that moves ions and proteins from in and out of the cell, or(3) a mediator of cell adhesion and cell-cell interaction. The cellularlocation of 103P2D6 can be assessed using subcellular fractionationtechniques widely used in cellular biology (see, e.g., Storrie B, et al.Methods Enzymol. 1990; 182:203–25). Prostate, colon, bladder, kidney orpancreas tumor cell lines are separated into nuclear, cytosolic, heavymembranes (lysosomes, peroxisomes, and mitochondria) and light membranes(plasma membrane and endoplasmic reticulum) fractions. The expression of103P2D6 is followed in each fraction by Western blotting. When 103P2D6participates in cell adhesion or cell-cell communication, 103P2D6 isused as a target for diagnostic, preventative and therapeutic purposes.

Example 15 Protein-Protein Interactions Mediated by 103P2D6

The determination of the specific proteins with which 103P2D6associates, including cytoskeleton and integrins, can be made, e.g.,using co-precipitation and Western blotting techniques (see, e.g.,Hamilton B J, et al. Biochem Biophys. Res. Commun. 1999, 261:646).Immunoprecipitates from cells expressing 103P2D6 and cells lacking103P2D6 are compared for specific protein-protein associations. 103P2D6also associates with effector molecules, such as C2-domain containingproteins. Studies comparing 103P2D6 positive and 103P2D6 negative cellsas well as studies comparing unstimulated/resting cells and cellstreated with epithelial cell activators, such as cytokines, androgen andantibodies reveal unique interactions. In addition, protein-proteininteractions can be studied using two yeast hybrid methodology (see,e.g., Curr Opin Chem Biol. 1999, 3:64). A vector carrying a library ofproteins fused to the activation domain of a transcription factor isintroduced into yeast expressing a 103P2D6-DNA-binding domain fusionprotein and a reporter construct. Protein-protein interaction isdetected by calorimetric reporter activity. Specific association witheffector molecules and transcription factors directs one of skill to themode of action of 103P2D6, and thus identifies therapeutic, preventativeand/or diagnostic targets for cancer.

Example 16 Regulation of Transcription by 103P2D6

The 103P2D6 protein contains a leucine zipper at its amino-terminus.Leucine zippers play a role in protein dimerization and determinesequence-specific DNA binding (Luscher B, Larsson L G. Oncogene. 1999;18:2955), and are therefore transcriptional regulators. Analogously,103P2D6 can regulate tumor progression by regulating gene expression.The role that 103P2D6 plays in regulating gene expression can beevaluated, e.g., by studying gene expression in cells expressing orlacking 103P2D6. For example, RNA from parental and 103P2D6-expressingNIH3T3 and PC3 cells is extracted and hybridized to commerciallyavailable gene arrays (Clontech). Resting cells as well as cells treatedwith serum, cytokines, androgen or antibodies are compared.Differentially expressed genes are identified and mapped to biologicalpathways. When 103P2D6 regulates transcription, 103P2D6 is used as atarget for diagnostic, preventative and therapeutic purposes.

Example 17 103P2D6 Monoclonal Antibody Mediated Inhibition of ProstateTumors in Vivo

The significant expression of 103P2D6, in cancer tissues, together withits restrictive expression in normal tissues makes 103P2D6 a target forantibody therapy. Similarly, 103P2D6 is a target for T cell-basedimmunotherapy. Thus, the therapeutic efficacy of anti-103P2D6 mAbs inhuman prostate cancer xenograft mouse models is evaluated by using theandrogen-dependent LAPC-9 xenograft (Craft, N., et al., Cancer Res,1999. 59(19): p. 5030–6) and the androgen independent recombinant cellline PC3-103P2D6 (see, e.g., Kaighn, M. E., et al., Invest Urol, 1979.17(1): p. 16–23).

Antibody efficacy on tumor growth and metastasis formation is studied,e.g., in a mouse orthotopic prostate cancer xenograft model. Thesestudies demonstrate that anti-103P2D6 MAbs inhibit formation of both theandrogen-dependent LAPC-9 and androgen-independent PC3-103P2D6 tumorxenografts. Anti-103P2D6 mAbs also retard the growth of establishedorthotopic tumors significantly and prolonged survival of tumor-bearingmice. These results indicate the utility of anti-103P2D6 mAbs in thetreatment of local and advanced stages of prostate cancer.

Administration of the anti-103P2D6 mAbs led to retardation ofestablished orthotopic tumor growth and inhibition of metastasis todistant sites, resulting in a significant prolongation in the survivalof tumor-bearing mice. These studies indicate that 103P2D6 as anattractive target for immunotherapy and demonstrate the therapeuticpotential of anti-103P2D6 mAbs for the treatment of local and metastaticprostate cancer. This example demonstrates that unconjugated 103P2D6monoclonal antibodies are effective to inhibit the growth of humanprostate tumor xenografts grown in SCID mice; accordingly a combinationof such efficacious monoclonal antibodies is also effective.

Tumor Inhibition Using Multiple Unconjugated 103P2D6 mAbs

Materials and Methods

103P2D6 Monoclonal Antibodies:

Monoclonal antibodies are raised against 103P2D6 as described in Example9.

Antibodies are characterized by ELISA, Western blot, FACS, andimmunoprecipitation for their capacity to bind 103P2D6. Epitope mappingdata for the anti-103P2D6 mAbs, as determined by ELISA and Westernanalysis, recognize epitopes on the 103P2D6 protein. Immunohistochemicalanalysis of prostate cancer tissues and cells with these antibodies isperformed.

Antibody Formulation:

The monoclonal antibodies are purified from hybridoma tissue culturesupernatants by Protein-G Sepharose chromatography, dialyzed againstPBS, filter sterilized, and stored at −20° C. Protein determinations areperformed by a Bradford assay (Bio-Rad, Hercules, California). Atherapeutic monoclonal antibody or a cocktail comprising a mixture ofindividual monoclonal antibodies is prepared and used for the treatmentof mice receiving subcutaneous or orthotopic injections of LAPC-9prostate tumor xenografts.

Prostate Cancer Xenografts and Cell Lines.

The LAPC-9 xenograft, which expresses a wild-type androgen receptor andproduces prostate-specific antigen (PSA), is passaged in 6- to8-week-old male ICR-severe combined immunodeficient (SCID) mice (TaconicFarms) by s.c. trocar implant (Craft, N., et al., supra). Single-cellsuspensions of LAPC-9 tumor cells are prepared as described in Craft, etal. The prostate carcinoma cell line PC3 (American Type CultureCollection) is maintained in DMEM supplemented with L-glutamine and 10%(vol/vol) FBS.

A PC3-103P2D6 cell population is generated by retroviral gene transferas described in Hubert, R. S., et al, Proc Natl Acad Sci U S A, 1999.96(25): p. 14523–8. Anti-103P2D6 staining is detected by using anFITC-conjugated goat anti-mouse antibody (Southern BiotechnologyAssociates) followed by analysis on a Coulter Epics-XL flow cytometer.

XenoUraft Mouse Models.

Subcutaneous (s.c.) tumors are generated by injection of 1×10⁶ LAPC-9,PC3, or PC3-103P2D6 cells mixed at a 1:1 dilution with Matrigel(Collaborative Research) in the right flank of male 30 SCID mice. Totest antibody efficacy on tumor formation, i.p. antibody injections arestarted on the same day as tumor-cell injections. As a control, mice areinjected with either purified mouse IgG (ICN) or PBS; or a purifiedmonoclonal antibody that recognizes an irrelevant antigen not expressedin human cells. In preliminary studies, no difference is found betweenmouse IgG or PBS on tumor growth. Tumor sizes are determined by verniercaliper measurements, and the tumor volume is calculated aslength×width×height. Mice with s.c. tumors greater than 1.5 cm indiameter are sacrificed. PSA levels are determined by using a PSA ELISAkit (Anogen, Mississauga, Ontario). Circulating levels of anti-103P2D6mAbs are determined by a capture ELISA kit (Bethyl Laboratories,Montgomery, Tex.).

Orthotopic injections are performed under anesthesia by usingketamine/xylazine. An incision is made through the abdominal muscles toexpose the bladder and seminal vesicles, which then are deliveredthrough the incision to expose the dorsal prostate. LAPC-9 cells (5×10⁵)mixed with Matrigel are injected into each dorsal lobe in a 10-μlvolume. To monitor tumor growth, mice are bled on a weekly basis fordetermination of PSA levels. Based on the PSA levels, the mice aresegregated into groups for the appropriate treatments. To test theeffect of anti-103P2D6 mAbs on established orthotopic tumors, i.p.antibody injections are started when PSA levels reach 2–80 ng/ml.

Anti-103P2D6 mAbs Inhibit Formation of 103P2D6-ExpressingProstate-Cancer Tumors

We next test the effect of anti-103P2D6 mAbs on tumor formation by usingthe LAPC-9 orthotopic model. As compared with the s.c. tumor model, theorthotopic model, which requires injection of tumor cells directly inthe mouse prostate, results in a local tumor growth, development ofmetastasis in distal sites, deterioration of mouse health, andsubsequent death (Saffran, D., et al., PNAS 10:1073–1078 orwww.pnas.org/cgi/doi/10.1073/pnas.051624698; Fu, X., et al., Int JCancer, 1992. 52(6): p. 987–90; Kubota, T., J Cell Biochem, 1994. 56(1):p. 4–8). The features make the orthotopic model more representative ofhuman disease progression and allowed us to follow the therapeuticeffect of mAbs on clinically relevant end points.

Accordingly, LAPC-9 tumor cells are injected into the mouse prostate,and 2 days later, the mice are segregated into two groups and treatedwith either 200 μg of 1G8 mAb or PBS three times per week for two weeks.Mice are monitored weekly for circulating PSA levels as an indicator oftumor growth.

A major advantage of the orthotopic prostate-cancer model is the abilityto study the development of metastases. Formation of metastasis in micebearing established orthotopic tumors is studies by IHC analysis on lungsections using an antibody against a prostate-specific cell-surfaceprotein STEAP expressed at high levels in LAPC-9 xenografts (Hubert, R.S., et al., Proc Natl Acad Sci U S A, 1999. 96(25): p. 14523–8).

Mice bearing established orthotopic LAPC-9 tumors are administered 11injections of either anti-103P2D6 mAb or PBS over a 4-week period. Micein both groups are allowed to establish a high tumor burden (PSA levelsgreater than 300 ng/ml), to ensure a high frequency of metastasisformation in mouse lungs. Mice then are killed and their prostate andlungs are analyzed for the presence of LAPC-9 cells by anti-STEAP IHCanalysis.

These studies demonstrate a broad anti-tumor efficacy of anti-103P2D6antibodies on initiation and progression of prostate cancer in xenograftmouse models. Anti-103P2D6 antibodies inhibit tumor formation of bothandrogen-dependent and androgen-independent tumors as well as retardingthe growth of already established tumors and prolong the survival oftreated mice. Moreover, anti-103P2D6 mAbs demonstrate a dramaticinhibitory effect on the spread of local prostate tumor to distal sites,even in the presence of a large tumor burden. Thus, anti-103P2D6 mAbsare efficacious on major clinically relevant end points/PSA levels(tumor growth), prolongation of survival, and health.

Throughout this application, various publications are referenced (withinparentheses for example). The disclosures of these publications arehereby incorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

TABLE I Cancer Tissues That Express 103P2D6 prostate colon stomach lungpancreas breast bladder bone kidney testis cervix ovary uterus

TABLE II AMINO ACID ABBREVIATIONS SINGLE LETTER THREE LETTER FULL NAME FPhe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cyscysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamineR Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asnasparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid EGlu glutamic acid G Gly glycine

TABLE III AMINO ACID SUBSTITUTION MATRIX Adapted from the GCG Software9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix).The higher the value, the more likely a substitution is found inrelated, natural proteins. A C D E F G H I K L M N P Q R S T V W Y 4 0−2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 A 9 −3 −4 −2 −3 −3 −1−3 −1 −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 C 6 2 −3 −1 −1 −3 −1 −4 −3 1 −1 0 −20 −1 −3 −4 −3 D 5 −3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3 −2 E 6 −3 −1 0−3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 F 6 −2 −4 −2 −4 −3 0 −2 −2 −2 0 −2 −3 −2−3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 H 4 −3 2 1 −3 −3 −3 −3 −2 −1 3−3 −1 I 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 K 4 2 −3 −3 −2 −2 −2 −1 1 −2 −1 L5 −2 −2 0 −1 −1 −1 1 −1 −1 M 6 −2 0 0 1 0 −3 −4 −2 N 7 −1 −2 −1 −1 −2 −4−3 P 5 1 0 −1 −2 −2 −1 Q 5 −1 −1 −3 −3 −2 R 4 1 −2 −3 −2 S 5 0 −2 −2 T 4−3 −1 V 11 2 W 7 Y

TABLE IV (A) HLA CLASS I SUPERMOTIFS SUPERMOTIF POSITION 2 C-TERMINUS A2L, I, V, M, A, T, Q L, I, V, M, A, T A3 A, V, I, L, M, S, T R, K B7 P A,L, I, M, V, F, W, Y B44 D, E F, W, Y, L, I, M, V, A A1 T, S, L, I, V, MF, W, Y A24 F, W, Y, L, V, I, M, T F, I, Y, W, L, M B27 R, H, K A, L, I,V, M, Y, F, W B58 A, S, T F, W, Y, L, I, V B62 L, V, M, P, I, Q F, W, Y,M, I, V

TABLE IV (B) HLA CLASS II SUPERMOTIF 1 6 9 W, F, Y, V, .I, L A, V, I, L,P, C, S, T A, V, I, L, C, S, T, M, Y

TABLE V Scoring Results 103P2D6 HLA peptides A1 9MERS Score (Estimate ofHalf Time Start Subsequence of Disassociation of a Molecule RankPosition Residue Listing Containing This Subsequence)  1  52 NAEQPELVF45.000   2 253 LTEAHGKWR 22.500   3 105 SMEAQGLSF 22.500   4 356DTDNPPLYC 6.250  5 153 STDGTFMPS 6.250  6 161 SIDVTNESR 5.000  7 321YLVPSLTRY 5.000  8 374 ALFPSLGTY 5.000  9 425 VLDIPTTQR 5.000 10 473LLDWQGIFA 2.500 11 500 CLFIFVLIY 2.500 12 208 TQDYKWVDR 1.500 13 538VSETSCQVS 1.350 14 450 YSEEIKSNI 1.350 15 203 IGLPNTQDY 1.250 16  69WTYSGQWMY 1.250 17 506 LIYVRVFRK 1.000 18 434 QTACGTVGK 1.000 19 378SLGTYDLEK 1.000 20 405 TLEAHQSKV 0.900 21 461 LHEASENLK 0.900 22 118LLEGNFSLC 0.900 23 126 CVENKNGSG 0.900 24  84 QAEVQNHST 0.900 25 520NSQPLNLAL 0.750 26  63 ASASTWWTY 0.750 27 222 WSGNDTCLY 0.750 28 233QNQTKGLLY 0.625 29  21 LVQPQHLLA 0.500 30 404 QTLEAHQSK 0.500 31 136FLGNIPKQY 0.500 32 496 IVLFCLFIF 0.500 33 291 SVNNSGLFF 0.500 34 383DLEKAILNI 0.450 35 394 AMEQEFSAT 0.450 36  55 QPELVFVPA 0.450 37 299FLCGNGVYK 0.400 38 313 WSGRCGLGY 0.375 39 442 KQCCLYINY 0.375 40 414SSLASASRK 0.300 41 413 VSSLASASR 0.300 42  71 YSGQWMYER 0.300 43 134GPFLGNIPK 0.250 44 354 QGDTDNPPL 0.250 45 174 DTSVCLGTR 0.250 46 224GNDTCLYSC 0.250 47 540 ETSCQVSNR 0.250 48 150 WFDSTDGTF 0.250 49 502FIFVLIYVR 0.200 50 505 VLIYVRVFR 0.200

TABLE VI Scoring Results 103P2D6 HLA peptides A1 10-MERS Score (Estimateof Half Time Start Subsequence of Disassociation of a Molecule RankPosition Residue Listing Containing This Subsequence)  1 473 LLDWQGIFAK50.000   2 394 AMEQEFSATK 18.000   3 354 QGDTDNPPLY 12.500   4 262CADASITNDK 10.000   5 483 VGDWFRSWGY 6.250  6 118 LLEGNFSLCV 4.500  7464 ASENLKNVPL 2.700  8 538 VSETSCQVSN 2.700  9 153 STDGTFMPSI 2.500 10356 DTDNPPLYCN 2.500 11 337 ITNLRSFIHK 2.500 12 499 FCLFIFVLIY 2.500 13451 SEEIKSNIQR 2.250 14 253 LTEAHGKWRC 2.250 15  46 LCEHLDNAEQ 1.800 16 52 NAEQPELVFV 1.800 17 450 YSEEIKSNIQ 1.350 18 438 GTVGKQCCLY 1.250 19505 VLIYVRVFRK 1.000 20  49 HLDNAEQPEL 1.000 21  84 QAEVQNHSTS 0.900 22405 TLEAHQSKVS 0.900 23 104 ASMEAQGLSF 0.750 24 290 LSVNNSGLFF 0.750 25203 IGLPNTQDYK 0.500 26 161 SIDVTNESRN 0.500 27 202 LIGLPNTQDY 0.500 28425 VLDIPTTQRQ 0.500 29 358 DNPPLYCNPK 0.500 30 373 RALFPSLGTY 0.500 31207 NTQDYKWVDR 0.500 32 495 LIVLFCLFIF 0.500 33  86 EVQNHSTSSY 0.500 34198 SAVPLIGLPN 0.500 35  55 QPELVFVPAS 0.450 36 105 SMEAQGLSFA 0.450 37542 SCQVSNRAMK 0.400 38 232 CQNQTKGLLY 0.375 39 413 VSSLASASRK 0.300 40520 NSQPLNLALS 0.300 41 213 WVDRNSGLTW 0.250 42 307 KGFPPKWSGR 0.250 43133 SGPFLGNIPK 0.250 44 461 LHEASENLKN 0.225 45 412 KVSSLASASR 0.200 46504 FVLIYVRVFR 0.200 47 460 RLHEASENLK 0.200 48 228 CLYSCQNQTK 0.200 49527 ALSPQQSAQL 0.200 50 377 PSLGTYDLEK 0.150

TABLE VII Scoring Results 103P2D6 HLA peptides A2 9MERS Score (Estimateof Half Time Start Subsequence of Disassociation of a Molecule RankPosition Residue Listing Containing This Subsequence)  1 497 VLFCLFIFV4571.688   2 117 RLLEGNFSL 1879.592   3 493 VLLIVLFCL 1792.489   4  45WLCEHLDNA 105.596   5 282 WLTGSNLTL 98.267  6 492 YVLLIVLFC 93.592  7 13 TLTAFLTIL 76.550  8  10 LQLTLTAFL 74.930  9  75 WMYERVWYP 61.634 10495 LIVLFCLFI 50.968 11  79 RVWYPQAEV 50.512 12 318 GLGYLVPSL 49.134 13329 YLTLNASQI 47.991 14 241 YQLFRNLFC 47.151 15 339 NLRSFIHKV 46.199 16239 LLYQLFRNL 44.160 17  11 QLTLTAFLT 43.222 18 460 RLHEASENL 42.917 19537 LVSETSCQV 42.418 20  20 ILVQPQHLL 36.316 21 228 CLYSCQNQT 23.846 22110 GLSFAQVRL 21.362 23 518 SLNSQPLNL 21.362 24 289 TLSVNNSGL 21.362 25393 KAMEQEFSA 21.249 26 473 LLDWQGIFA 18.580 27 535 QLLVSETSC 18.382 28 8 ALLQLTLTA 18.382 29 292 VNNSGLFFL 17.393 30  53 AEQPELVFV 17.108 31 27 LLAPVFRTL 13.800 32 108 AQGLSFAQV 13.398 33  26 HLLAPVFRT 12.506 34 19 TILVQPQHL 10.868 35 119 LEGNFSLCV  8.737 36 336 QITNLRSFI  7.890 37219 GLTWSGNDT  7.452 38 552 GLTTHQYDT  7.452 39 246 NLFCSYGLT  5.377 40405 TLEAHQSKV  4.451 41 242 QLFRNLFCS  3.678 42 238 GLLYQLFRN  3.678 43156 GTFMPSIDV  3.574 44 322 LVPSLTRYL  3.495 45  38 LTNQSNCWL  2.774 46 36 SILTNQSNC  2.527 47 387 AILNISKAM  2.527 48 499 FCLFIFVLI  2.202 49106 MEAQGLSFA  2.077 50 490 WGYVLLIVL  1.936

TABLE VIII Scoring Results 103P2D6 HLA peptides A2 10-MERS Score(Estimate of Half Time Start Subsequence of Disassociation of a MoleculeRank Position Residue Listing Containing This Subsequence)  1 500CLFIFVLIYV 3255.381   2 497 VLFCLFIFVL 2792.698   3 492 YVLLIVLFCL424.398   4 496 IVLFCLFIFV 400.023   5 494 LLIVLFCLFI 370.677   6 296GLFFLCGNGV 257.342   7 502 FIFVLIYVRV 244.154   8  37 ILTNQSNCWL199.738   9  9 LLQLTLTAFL 199.738  10 117 RLLEGNFSLC 143.193  11 291SVNNSGLFFL 137.144  12 136 FLGNIPKQYC 125.690  13 536 LLVSETSCQV118.238  14 321 YLVPSLTRYL 108.094  15  13 TLTAFLTILV 69.552 16 148ILWFDSTDGT 51.522 17 331 TLNASQITNL 49.134 18  3 SLSNCALLQL 49.134 19238 GLLYQLFRNL 30.036 20 118 LLEGNFSLCV 28.756 21  22 VQPQHLLAPV 27.57322  10 LQLTLTAFLT 27.564 23 527 ALSPQQSAQL 21.362 24  11 QLTLTAFLTI19.822 25 445 CLYINYSEEI 16.359 26 338 TNLRSFIHKV 14.682 27 404QTLEAHQSKV 14.654 28 329 YLTLNASQIT 14.054 29 518 SLNSQPLNLA 11.426 30 19 TILVQPQHLL 10.868 31 393 KAMEQEFSAT 10.441 32 110 GLSFAQVRLL  9.82733 472 PLLDWQGIFA  9.119 34 353 TQGDTDNPPL  8.880 35  20 ILVQPQHLLA 8.446 36 318 GLGYLVPSLT  7.452 37  12 LTLTAFLTIL  6.687 38 205LPNTQDYKWV  6.368 39 335 SQITNLRSFI  5.818 40 378 SLGTYDLEKA  5.599 41439 TVGKQCCLYI  5.021 42 234 NQTKGLLYQL  4.982 43 325 SLTRYLTLNA  4.96844 525 NLALSPQQSA  4.968 45 544 QVSNRAMKGL  4.299 46 220 LTWSGNDTCL 4.186 47  58 LVFVPASAST  4.101 48 488 RSWGYVLLIV  3.554 49  61VPASASTWWT  3.411 50 343 FIHKVTPHRC  3.142

TABLE IX Scoring Results 103P2D6 HLA PEPTIDES A3 9-MERS Score (Estimateof Half Time Start Subsequence of Disassociation of a Molecule RankPosition Residue Listing Containing This Subsequence)  1 500 CLFIFVLIY360.000   2 378 SLGTYDLEK 120.000   3 304 GVYKGFPPK 90.000  4 506LIYVRVFRK 90.000  5 204 GLPNTQDYK 60.000  6 299 FLCGNGVYK 30.000  7 321YLVPSLTRY 13.500  8 374 ALFPSLGTY 13.500  9 505 VLIYVRVFR  9.000 10 494LLIVLFCLF  9.000 11 502 FIFVLIYVR  9.000 12 493 VLLIVLFCL  6.075 13 425VLDIPTTQR  6.000 14 134 GPFLGNIPK  6.000 15 497 VLFCLFIFV  6.000 16 318GLGYLVPSL  5.400 17 117 RLLEGNFSL  4.050 18 178 CLGTRQCSR  4.000 19 105SMEAQGLSF  4.000 20 136 FLGNIPKQY  3.000 21  69 WTYSGQWMY  3.000 22  9LLQLTLTAF  3.000 23  13 TLTAFLTIL  2.700 24 404 QTLEAHQSK  2.250 25  26HLLAPVFRT  2.025 26 447 YINYSEEIK  2.000 27 110 GLSFAQVRL  1.800 28 282WLTGSNLTL  1.800 29  20 ILVQPQHLL  1.350 30  75 WMYERVWYP  1.350 31 496IVLFCLFIF  1.350 32 518 SLNSQPLNL  1.200 33 434 QTACGTVGK  1.000 34 552GLTTHQYDT  0.900 35 118 LLEGNFSLC  0.900 36 339 NLRSFIHKV  0.900 37 239LLYQLFRNL  0.900 38 460 RLHEASENL  0.900 39 543 CQVSNRAMK  0.900 40 242QLFRNLFCS  0.900 41 442 KQCCLYINY  0.720 42 289 TLSVNNSGL  0.600 43 359NPPLYCNPK  0.600 44 343 FIHKVTPHR  0.600 45 329 YLTLNASQI  0.600 46 508YVRVFRKSR  0.600 47  8 ALLQLTLTA  0.600 48 495 LIVLFCLFI  0.540 49 383DLEKAILNI  0.540 50 228 CLYSCQNQT  0.500

TABLE X Scoring Results 103P2D6 HLA peptides A3 10-MERS Score (Estimateof Half Time Start Subsequence of Disassociation of a Molecule RankPosition Residue Listing Containing This Subsequence)  1 505 VLIYVRVFRK270.000   2 228 CLYSCQNQTK 100.000   3 473 LLDWQGIFAK 90.000  4 394AMEQEFSATK 60.000  5 242 QLFRNLFCSY 60.000  6 497 VLFCLFIFVL 40.500  7460 RLHEASENLK 30.000  8 239 LLYQLFRNLF 30.000  9 252 GLTEAHGKWR  9.00010 493 VLLIVLFCLF  9.000 11 445 CLYINYSEEI  9.000 12 337 ITNLRSFIHK 6.000 13 549 AMKGLTTHQY  6.000 14 494 LLIVLFCLFI  5.400 15 500CLFIFVLIYV  4.500 16  8 ALLQLTLTAF  4.500 17 478 GIFAKVGDWF  4.500 18 11 QLTLTAFLTI  3.600 19 296 GLFFLCGNGV  3.000 20 142 KQYCNQILWF  2.70021 178 CLGTRQCSRF  2.000 22 121 GNFSLCVENK  1.800 23  73 GQWMYERVWY 1.800 24 118 LLEGNFSLCV  1.800 25  3 SLSNCALLQL  1.800 26 403KQTLEAHQSK  1.800 27 204 GLPNTQDYKW  1.800 28 495 LIVLFCLFIF  1.350 29117 RLLEGNFSLC  1.350 30 438 GTVGKQCCLY  1.350 31 412 KVSSLASASR  1.20032 499 FCLFIFVLIY  1.080 33 331 TLNASQITNL  0.900 34 304 GVYKGFPPKW 0.900 35 527 ALSPQQSAQL  0.900 36 504 FVLIYVRVFR  0.900 37 467NLKNVPLLDW  0.900 38 424 HVLDIPTTQR  0.900 39 238 GLLYQLFRNL  0.810 40 75 WMYERVWYPQ  0.675 41 492 YVLLIVLFCL  0.608 42  49 HLDNAEQPEL  0.60043 219 GLTWSGNDTC  0.600 44 246 NLFCSYGLTE  0.600 45  20 ILVQPQHLLA 0.600 46  9 LLQLTLTAFL  0.600 47 289 TLSVNNSGLF  0.600 48  37ILTNQSNCWL  0.600 49 433 RQTACGTVGK  0.600 50 110 GLSFAQVRLL  0.540

TABLE XI Scoring Results 103P2D6 HLA peptides A11 9 MERS Score (Estimateof Half Time Start Subsequence of Disassociation of a Molecule RankPosition Residue Listing Containing This Subsequence)  1 304 GVYKGFPPK12.000   2 134 GPFLGNIPK 2.400  3 506 LIYVRVFRK 2.400  4 404 QTLEAHQSK1.500  5 204 GLPNTQDYK 1.200  6 434 QTACGTVGK 1.000  7 543 CQVSNRAMK0.900  8 378 SLGTYDLEK 0.800  9 320 GYLVPSLTR 0.720 10 229 LYSCQNQTK0.400 11 447 YINYSEEIK 0.400 12 299 FLCGNGVYK 0.400 13 502 FIFVLIYVR0.320 14 122 NFSLCVENK 0.200 15 508 YVRVFRKSR 0.200 16 359 NPPLYCNPK0.200 17 338 TNLRSFIHK 0.120 18 474 LDWQGIFAK 0.120 19 156 GTFMPSIDV0.120 20 505 VLIYVRVFR 0.120 21 208 TQDYKWVDR 0.120 22 308 GFPPKWSGR0.120 23  79 RVWYPQAEV 0.120 24 253 LTEAHGKWR 0.100 25 496 IVLFCLFIF0.090 26 178 CLGTRQCSR 0.080 27 425 VLDIPTTQR 0.080 28 343 FIHKVTPHR0.080 29 161 SIDVTNESR 0.080 30 480 FAKVGDWFR 0.080 31 142 KQYCNQILW0.072 32 540 ETSCQVSNR 0.060 33 395 MEQEFSATK 0.060 34 174 DTSVCLGTR0.060 35 117 RLLEGNFSL 0.054 36 438 GTVGKQCCL 0.045 37 365 NPKDNSTIR0.040 38  21 LVQPQHLLA 0.040 39  69 WTYSGQWMY 0.040 40 291 SVNNSGLFF0.040 41  89 NHSTSSYRK 0.040 42 333 NASQITNLR 0.040 43 237 KGLLYQLFR0.036 44 393 KAMEQEFSA 0.036 45 442 KQCCLYINY 0.036 46 251 YGLTEAHGK0.030 47 414 SSLASASRK 0.030 48  12 LTLTAFLTI 0.030 49 337 ITNLRSFIH0.030 50 380 GTYDLEKAI 0.030

TABLE XII Scoring Results 103P2D6 HLA peptides A11 10-MERS Score(Estimate of Half Time Start Subsequence of Disassociation of a MoleculeRank Position Residue Listing Containing This Subsequence)  1 337ITNLRSFIHK 2.000  2 505 VLIYVRVFRK 1.800  3 433 RQTACGTVGK 1.800  4 403KQTLEAHQSK 1.800  5 473 LLDWQGIFAK 1.200  6 412 KVSSLASASR 1.200  7 460RLHEASENLK 1.200  8 228 CLYSCQNQTK 0.800  9 504 FVLIYVRVFR 0.600 10 446LYINYSEEIK 0.600 11 424 HVLDIPTTQR 0.600 12 508 YVRVFRKSRR 0.400 13 250SYGLTEAHGK 0.400 14 394 AMEQEFSATK 0.400 15 298 FFLCGNGVYK 0.300 16 121GNFSLCVENK 0.240 17 542 SCQVSNRAMK 0.200 18 262 CADASITNDK 0.200 19 207NTQDYKWVDR 0.200 20  70 TYSGQWMYER 0.160 21  87 VQNHSTSSYR 0.120 22 304GVYKGFPPKW 0.120 23 108 AQGLSFAQVR 0.120 24 384 LEKAILNISK 0.120 25 252GLTEAHGKWR 0.120 26 501 LFIFVLIYVR 0.120 27 492 YVLLIVLFCL 0.090 28 479IFAKVGDWFR 0.080 29  88 QNHSTSSYRK 0.080 30 142 KQYCNQILWF 0.072 31 193RTWNSSAVPL 0.060 32 291 SVNNSGLFFL 0.060 33  97 KVTWHWEASM 0.060 34 380GTYDLEKAIL 0.060 35 177 VCLGTRQCSR 0.060 36 496 IVLFCLFIFV 0.060 37 507IYVRVFRKSR 0.060 38 342 SFIHKVTPHR 0.060 39 438 GTVGKQCCLY 0.045 40 439TVGKQCCLYI 0.040 41 213 WVDRNSGLTW 0.040 42 133 SGPFLGNIPK 0.040 43  73GQWMYERVWY 0.036 44 303 NGVYKGFPPK 0.030 45 203 IGLPNTQDYK 0.030 46 307KGFPPKWSGR 0.024 47 478 GIFAKVGDWF 0.024 48 451 SEEIKSNIQR 0.024 49 204GLPNTQDYKW 0.024 50 296 GLFFLCGNGV 0.024

TABLE XIII Scoring Results 103P2D6 HLA peptides A24 9-MERS Score(Estimate of Half Time Start Subsequence of Disassociation of a MoleculeRank Position Residue Listing Containing This Subsequence)  1 381TYDLEKAIL 200.000   2 491 GYVLLIVLF 180.000   3 240 LYQLFRNLF 180.000  4 143 QYCNQILWF 100.000   5 446 LYINYSEEI 82.500  6 398 EFSATKQTL24.000  7 486 WFRSWGYVL 20.000  8 498 LFCLFIFVL 20.000  9 511 VFRKSRRSL20.000 10 212 KWVDRNSGL 14.400 11 117 RLLEGNFSL 14.400 12 479 IFAKVGDWF14.000 13 507 IYVRVFRKS 13.860 14 245 RNLFCSYGL 12.000 15 150 WFDSTDGTF10.000 16 460 RLHEASENL  9.600 17 520 NSQPLNLAL  8.640 18 493 VLLIVLFCL 8.400 19  27 LLAPVFRTL  8.064 20  81 WYPQAEVQN  7.500 21 529 SPQQSAQLL 7.200 22  10 LQLTLTAFL  7.200 23 322 LVPSLTRYL  7.200 24  19 TILVQPQHL 7.200 25  94 SYRKVTWHW  7.000 26 210 DYKWVDRNS  7.000 27  38 LTNQSNCWL 6.000 28 449 NYSEEIKSN  6.000 29  20 ILVQPQHLL  6.000 30 545 VSNRAMKGL 6.000 31  4 LSNCALLQL  6.000 32 292 VNNSGLFFL  6.000 33 231 SCQNQTKGL 6.000 34 528 LSPQQSAQL  6.000 35  2 GSLSNCALL  6.000 36 518 SLNSQPLNL 6.000 37 438 GTVGKQCCL  6.000 38 232 CQNQTKGLL  6.000 39 466 ENLKNVPLL 6.000 40 194 TWNSSAVPL  6.000 41 371 TIRALFPSL  5.760 42 239 LLYQLFRNL 5.760 43 305 VYKGFPPKW  5.500 44 362 LYCNPKDNS  5.000 45  6 NCALLQLTL 4.800 46 235 QTKGLLYQL  4.800 47  42 SNCWLCEHL  4.800 48 103 EASMEAQGL 4.800 49 490 WGYVLLIVL  4.800 50 318 GLGYLVPSL  4.800

TABLE XIV Scoring Results 103P2D6 HLA peptides A24 10-MERS Score(Estimate of Half Time Start Subsequence of Disassociation of a MoleculeRank Position Residue Listing Containing This Subsequence)  1 328RYLTLNASQI 150.000   2 449 NYSEEIKSNI 84.000  3 375 LFPSLGTYDL 30.000  4486 WFRSWGYVLL 20.000  5 503 IFVLIYVRVF 15.000  6 517 RSLNSQPLNL 12.000 7 491 GYVLLIVLFC 10.500  8  26 HLLAPVFRTL 10.080  9 370 STIRALFPSL 8.640 10 238 GLLYQLFRNL  8.640 11 321 YLVPSLTRYL  8.640 12 498LFCLFIFVLI  8.400 13 492 YVLLIVLFCL  8.400 14 510 RVFRKSRRSL  8.000 15193 RTWNSSAVPL  8.000 16 240 LYQLFRNLFC  7.500 17 320 GYLVPSLTRY  7.50018  76 MYERVWYPQA  7.500 19  41 QSNCWLCEHL  7.200 20  18 LTILVQPQHL 7.200 21 528 LSPQQSAQLL  7.200 22  9 LLQLTLTAFL  7.200 23 317CGLGYLVPSL  7.200 24 109 QGLSFAQVRL  6.000 25  29 APVFRTLSIL  6.000 26331 TLNASQITNL  6.000 27 464 ASENLKNVPL  6.000 28 288 LTLSVNNSGL  6.00029 231 SCQNQTKGLL  6.000 30 281 WWLTGSNLTL  6.000 31 362 LYCNPKDNST 6.000 32 291 SVNNSGLFFL  6.000 33  12 LTLTAFLTIL  6.000 34  19TILVQPQHLL  6.000 35 381 TYDLEKAILN  5.000 36 305 VYKGFPPKWS  5.000 37489 SWGYVLLIVL  4.800 38 234 NQTKGLLYQL  4.800 39 380 GTYDlEKAIL  4.80040 519 LNSQpLNLAL  4.800 41 140 IPKQyCNQIL  4.800 42  5 SNCAlLQLTL 4.800 43 353 TQGDtDNPPL  4.800 44 527 ALSPqQSAQL  4.800 45  49HLDNaEQPEL  4.400 46 493 VLLIvLFCLF  4.320 47 544 QVSNrAMKGL  4.000 48323 VPSLtRYLTL  4.000 49  1 MGSLsNCALL  4.000 50 485 DWFRsWGYVL  4.000

TABLE XV Scoring Results 103P2D6 HLA peptides B7 9-MERS Score (Estimateof Half Time Start Subsequence of Disassociation of a Molecule RankPosition Residue Listing Containing This Subsequence)  1 376 FPSLGTYDL80.000   2 529 SPQQSAQLL 80.000   3 371 TIRALFPSL 40.000   4 314SGRCGLGYL 40.000   5  29 APVFRTLSI 24.000   6 322 LVPSLTRYL 20.000   7103 EASMEAQGL 12.000   8 418 SASRKDHVL 12.000   9 471 VPLLDWQGI 8.000 10140 IPKQYCNQI 8.000 11  20 ILVQPQHLL 6.000 12 197 SSAVPLIGL 6.000 13 275GHRTPTWWL 6.000 14 511 VFRKSRRSL 6.000 15 235 QTKGLLYQL 4.000 16 453EIKSNIQRL 4.000 17 110 GLSFAQVRL 4.000 18 493 VLLIVLFCL 4.000 19 486WFRSWGYVL 4.000 20 282 WLTGSNLTL 4.000 21  4 LSNCALLQL 4.000 22 545VSNRAMKGL 4.000 23 520 NSQPLNLAL 4.000 24  42 SNCWLCEHL 4.000 25 231SCQNQTKGL 4.000 26 239 LLYQLFRNL 4.000 27 528 LSPQQSAQL 4.000 28  13TLTAFLTIL 4.000 29  10 LQLTLTAFL 4.000 30 490 WGYVLLIVL 4.000 31  27LLAPVFRTL 4.000 32 111 LSFAQVRLL 4.000 33 466 ENLKNVPLL 4.000 34 518SLNSQPLNL 4.000 35  2 GSLSNCALL 4.000 36  19 TILVQPQHL 4.000 37  6NCALLQLTL 4.000 38 318 GLGYLVPSL 4.000 39 332 LNASQITNL 4.000 40 460RLHEASENL 4.000 41  1 MGSLSNCAL 4.000 42 289 TLSVNNSGL 4.000 43  23QPQHLLAPV 4.000 44 438 GTVGKQCCL 4.000 45 232 CQNQTKGLL 4.000 46 245RNLFCSYGL 4.000 47  38 LTNQSNCWL 4.000 48 117 RLLEGNFSL 4.000 49 292VNNSGLFFL 4.000 50 554 TTHQYDTSL 4.000

TABLE XVI Scoring Results 103P2D6 HLA peptides B7 10-MERS Score(Estimate of Half Time Start Subsequence of Disassociation of a MoleculeRank Position Residue Listing Containing This Subsequence)  1  29APVFRTLSIL 240.000   2 140 IPKQYCNQIL 80.000  3 323 VPSLTRYLTL 80.000  4510 RVFRKSRRSL 30.000  5 492 YYLLIVLFCL 20.000  6 291 SVNNSGLFFL 20.000 7 544 QVSNRAMKGL 20.000  8 310 PPKWSGRCGL 12.000  9 417 ASASRKDHVL12.000 10 527 ALSPQQSAQL 12.000 11 407 EAHQSKVSSL 12.000 12 419ASRKDHVLDI 12.000 13 196 NSSAVPLIGL  6.000 14 274 DGHRTPTWWL  6.000 15 19 TILVQPQHLL  6.000 16  97 KVTWHWEASM  5.000 17 109 QGLSFAQVRL  4.00018 220 LTWSGNDTCL  4.000 19 528 LSPQQSAQLL  4.000 20 331 TLNASQITNL 4.000 21 370 STIRALFPSL  4.000 22 437 CGTVGKQCCL  4.000 23 110GLSFAQVRLL  4.000 24 231 SCQNQTKGLL  4.000 25 353 TQGDTDNPPL  4.000 26193 RTWNSSAVPL  4.000 27 517 RSLNSQPLNL  4.000 28  41 QSNCWLCEHL  4.00029 519 LNSQPLNLAL  4.000 30 238 GLLYQLFRNL  4.000 31  26 HLLAPVFRTL 4.000 32 234 NQTKGLLYQL  4.000 33 205 LPNTQDYKWV  4.000 34 128ENKNGSGPFL  4.000 35 486 WFRSWGYVLL  4.000 36  12 LTLTAFLTIL  4.000 37529 SPQQSAQLLV  4.000 38 553 LTTHQYDTSL  4.000 39 317 CGLGYLVPSL  4.00040 288 LTLSVNNSGL  4.000 41 380 GTYDLEKAIL  4.000 42  18 LTILVQPQHL 4.000 43 554 TTHQYDTSLL  4.000 44  37 ILTNQSNCWL  4.000 45 515SRRSLNSQPL  4.000 46  9 LLQLTLTAFL  4.000 47 321 YLVPSLTRYL  4.000 48497 VLFCLFIFVL  4.000 49  5 SNCALLQLTL  4.000 50  3 SLSNCALLQL  4.000

TABLE XVII Scoring Results 103P2D6 HLA peptides B35 9-MERS Score(Estimate of Half Time Start Subsequence of Disassociation of a MoleculeRank Position Residue Listing Containing This Subsequence)  1 140IPKQYCNQI 24.000  2 376 FPSLGTYDL 20.000  3 529 SPQQSAQLL 20.000  4 222WSGNDTCLY 15.000  5 391 ISKAMEQEF 15.000  6 471 VPLLDWQGI 12.000  7  63ASASTWWTY 10.000  8 313 WSGRCGLGY 10.000  9  61 VPASASTWW 10.000 10 205LPNTQDYKW 10.000 11  29 APVFRTLSI  8.000 12 528 LSPQQSAQL  5.000 13 520NSQPLNLAL  5.000 14 545 VSNRAMKGL  5.000 15 197 SSAVPLIGL  5.000 16 111LSFAQVRLL  5.000 17  2 GSLSNCALL  5.000 18 290 LSVNNSGLF  5.000 19  4LSNGALLQL  5.000 20 103 EASMEAQGL  4.500 21  23 QPQHLLAPV  4.000 22 117RLLEGNFSL  4.000 23 460 RLHEASENL  4.000 24 442 KQCCLYINY  4.000 25 488RSWGYVLLI  4.000 26 314 SGRCGLGYL  3.000 27 235 QTKGLLYQL  3.000 28 453EIKSNIQRL  3.000 29 418 SASRKDHVL  3.000 30 128 ENKNGSGPF  3.000 31 371TIRALFPSL  3.000 32 115 QVRLLEGNF  3.000 33  92 TSSYRKVTW  2.500 34 167ESRNDDDDT  2.250 35  69 WTYSGQWMY  2.000 36 482 KVGDWFRSW  2.000 37 200VPLIGLPNT  2.000 38 309 FPPKWSGRC  2.000 39 323 VPSLTRYLT  2.000 40 203IGLPNTQDY  2.000 41 136 FLGNIPKQY  2.000 42 500 CLFIFVLIY  2.000 43 387AILNISKAM  2.000 44 245 RNLFCSYGL  2.000 45 439 TVGKQCCLY  2.000 46 321YLVPSLTRY  2.000 47 428 IPTTQRQTA  2.000 48 132 GSGPFLGNI  2.000 49  98VTWHWEASM  2.000 50 278 TPTWWLTGS  2.000

TABLE XVIII Scoring Results 103P2D6 HLA peptides A35 10-MERS Score(Estimate of Half Time Start Subsequence of Disassociation of a MoleculeRank Position Residue Listing Containing This Subsequence)  1 140IPKQYCNQIL 60.000  2 323 VPSLTRYLTL 20.000  3  23 QPQHLLAPVF 20.000  4471 VPLLDWQGIF 20.000  5  29 APVTRTLSIL 20.000  6 365 NPKDNSTIRA 12.000 7 386 KAILNISKAM 12.000  8 373 RALFPSLGTY 12.000  9 517 RSLNSQPLNL10.000 10 104 ASMEAQGLSF 10.000 11 541 TSCQVSNRAM 10.000 12 549AMKGLTTHQY  6.000 13 310 PPKWSGRCGL  6.000 14 205 LPNTQDYKWV  6.000 15419 ASRKDHVLDI  6.000 16 528 LSPQQSAQLL  5.000 17 230 YSCQNQTKGL  5.00018 334 ASQITNLRSF  5.000 19 313 WSGRCGLGYL  5.000 20  41 QSNCWLCEHL 5.000 21 417 ASASRKDHVL  5.000 22 196 NSSAVPLIGL  5.000 23 290LSVNNSGLFF  5.000 24 529 SPQQSAQLLV  4.000 25  97 KVTWHWEASM  4.000 26353 TQGDTDNPPL  3.000 27 235 QTKGLLYQLF  3.000 28 128 ENKNGSGPFL  3.00029 380 GTYDLEKAIL  3.000 30 407 EAHQSKVSSL  3.000 31  73 GQWMYERVWY 3.000 32  93 SSYRKVTWHW  2.500 33 391 ISKAMEQEFS  2.250 34 167ESRNDDDDTS  2.250 35 438 GTVGKQCCLY  2.000 36  82 YPQAEVQNHS  2.000 37232 CQNQTKGLLY  2.000 38 242 QLFRNLFCSY  2.000 39  61 VPASASTWWT  2.00040 510 RVFRKSRRSL  2.000 41  51 DNAEQPELVF  2.000 42 159 MPSIDVTNES 2.000 43 348 TPHRCTQGDT  2.000 44 488 RSWGYVLLIV  2.000 45 278TPTWWLTGSN  2.000 46 142 KQYCNQILWF  2.000 47 193 RTWNSSAVPL  2.000 48 86 EVQNHSTSSY  2.000 49 202 LIGLPNTQDY  2.000 50 499 FCLFIFVLIY  2.000

TABLE XIX Motif-bearing Subsequences of the 103P2D6 ProteinPost-translational modification Sites 15 N-glycosylation sites  1  40–43NQSN  2  89–92 NHST  3 122–125 NFSL  4 131–134 NGSG  5 166–169 NESR  6192–195 NRTW  7 196–199 NSSA  8 225–228 NDTC  9 234–237 NQTK 10 287–290NLTL 11 293–296 NNSG 12 333–336 NASQ 13 369–372 NSTI 14 390–393 NISK 15449–452 NYSE One cAMP- and cGMP-dependent protein kinase phosphorylationsite  1  96–99 RKVT Eight protein kinase C phosphorylation sites  1 94–96 SYR  2 191–193 TNR  3 314–316 SGR  4 371–373 TIR  5 420–422 SRK 6 431–433 TQR  7 515–517 SRR  8 546–548 SNR Significance of Posttranslational modifications Four casein kinase II phosphorylation site 1 168–171 SRND  2 223–226 SGND  3 353–356 TQGD  4 420–423 SRKD NineN-myristoylation sites  1   2–7 GSLSNC  2 110–115 GLSFAQ  3 180–185GTRQCS  4 204–209 GLPNTQ  5 219–224 GLTWSG  6 224–229 GNDTCL  7 252–257GLTEAH  8 285–290 GSNLTL  9 304–309 GVYKGF

Glycosylation, or attachment of carbohydrate chains to asparagines,serine or threonine moieties, plays an important role in proteinfolding, stability and protection from degradation (Biochem J. 2000,348:1). In addition, glycosylation allows sorting of proteins from theendoplasmic reticulum along the secretory pathway, thereby contributingto protein localization (FEBS Lett. 2000, 476:32). Also, glycosylationoften contributes to cell adhesion and immune recognition (J Mol Biol.1999, 293:351).

Phosphorylation, whether by PKC, cAMP and c-GMP dependent kinases, orcasein kinases, exhorts a profound effect on proteins. Phosphorylationmediates protein-protein interactions as well as signaling pathwayactivation. Phosphorylation also controls protein localization,translocation and enzymatic activity, and regulates transcriptionalactivation (Mol Immunol. 2000, 37: 1; Cell Mol Life Sci. 2000,57:1172–83). By means of post-translational modification of protein,phosphorylation regulates cellular functions, including proliferation,migration and gene expression (Cell Prolif. 2000, 33:341; Mol Biol Cell.2001, 12:351).

Myristoylation serves to anchor numerous proteins to the cytoplasmicface of the plasma membrane. This process serves to facilitate proteinrecruitment, complex assembly and signaling through proteins (CurrentOpinion Cell Biol. 1994, 6:219; J Biol Chem. 1996, 271:1573).

Motifs Found in 103P2D6

-   14–35 Leucine zipper pattern LTAFLTILVQPQHLLAPVFRTL 484–516    Large-conductance mechanosensitive channel,    GDWFRSWGYVLLIVLFCLFEFVLIYVRVFRKSRR-   487–507 Sodium/chloride neurotransmitter symporter signature    FRSWGYVLLIVLFCLFIFVLI    Topology and Transmembrane Domains

Using three different prediction programs, 103P2D6 is proposed to be amembrane associated protein, primarily expressed at the cell surface(64%). There is a possibility that 103P2D6 is associated with theendoplasmic reticulum (21%).

Several Possibilities for Transmembrane Domains and Topologies:

-   PSORT (http://psort.nibb.ac.jp) indicates the presence of 1 TM    domain at aa 493–509 (VLLIVLFCLFIFVLIYV), and 1 signal sequence at    aa 1–24 (MGSLSNCALLQLTLTAFLTILVQP), with a cleavage site between aa    24 and aa 25.-   TMpred (www.ch.3mbnet.org) indicates that 103P2D6 may have four    transmembrane domains, which are listed below:-   TM1 aa 4–22-o-i-   TM2 aa 58–77-i-o-   TM3 aa 283–300-o-i-   TM4 aa 493–509-i-o

Although this scenario is less likely to occur, in view of thesimilarity with envelope protein which has a single transmembranedomain, it is possible that 103P2D6 exhibits a multiple TM configuration

-   In all cases, the N-terminus extends outside the cell, while the    C-terminus is intracellular.

TABLE XX Frequently Occurring Motifs avg. % Name identity DescriptionPotential Function zf-C2H2 34% Zinc finger, Nucleic acid-binding C2H2type protein functions as transcription factor, nuclear locationprobable cytochrome_b_N 68% Cytochrome b(N- membrane boundterminal)/b6/petB oxidase, generate superoxide ig 19% Immunoglobulindomains are one domain hundred amino acids long and include a conservedintradomain disulfide bond. WD40 18% WD domain, tandem repeats of G-betarepeat about 40 residues, each containing a Trp-Asp motif. Function insignal transduction and protein interaction PDZ 23% PDZ domain mayfunction in targeting signaling molecules to sub- membranous sites LRR28% Leucine Rich short sequence motifs Repeat involved in protein-protein interactions pkinase 23% Protein kinase conserved catalyticdomain core common to both serine/threonine and tyrosine protein kinasescontaining an ATP binding site and a catalytic site PH 16% PH domainpleckstrin homology involved in intra- cellular signaling or asconstituents of the cytoskeleton EGF 34% EGF-like domain 30–40amino-acid long found in the extra- cellular domain of membrane-boundproteins or in secreted proteins rvt 49% Reverse transcriptase(RNA-dependent DNA polymerase) ank 25% Ank repeat Cytoplasmic protein,associates integral membrane proteins to the cytoskeleton oxidored_q132% NADH- membrane associated. Ubiquinone/ Involved in protonplastoquinone translocation across (complex I), the membrane variouschains efhand 24% EF hand calcium-binding domain, consists of a12residue loop flanked on both side by a 12 residue alpha- helical domainrvp 79% Retroviral Aspartyl or acid aspartyl protease proteases,centered on a catalytic aspartyl residue Collagen 42% Collagen tripleextracellular structural helix repeat proteins involved (20 copies) information of connective tissue. The sequence consists of the G-X-Y andthe polypeptide chains forms a triple helix. fn3 20% Fibronectin typeLocated in the extra- III domain cellular ligand-binding region ofreceptors and is about 200 amino acid residues long with two pairs ofcysteines involved in disulfide bonds 7tm_1 19% 7 transmembrane sevenhydrophobic receptor transmembrane (rhofopsin regions, with the family)N-terminus located extracellularly while the C-terminus is cytoplasmic.Signal through G proteins

1. An isolated polynucleotide, comprising the nucleotide sequence shownin FIG. 2 (SEQ ID NO: 1), from nucleotide residue number 805 throughnucleotide residue number 2493, wherein T can also be U, or apolynucleotide that is fully complementary thereto; or a polynucleotidecomprising the sequence of a cDNA contained in the plasmid designatedp103P2D6-B deposited with American Type Culture Collection as AccessionNo. PTA-1895; and a polynucleotide comprising the sequence of a cDNAcontained in the plasmid designated p103P2D6-2 deposited with AmericanType Culture Collection as Accession No. PTA-1155; and, a polynucleotidethat encodes the amino acid sequence of SEQ ID NO:
 2. 2. Thepolynucleotide of claim 1 comprising the nucleic acid sequence shown inFIG. 2 (SEQ ID NO: 1), wherein T can also be U.
 3. The polynucleotidethat is fully complementary to the polynucleotide of claim
 2. 4. Thepolynucleotide of claim 1 comprising the sequence shown in FIG. 2 (SEQID NO: 1), from nucleotide residue number 805 through nucleotide residuenumber 2493, wherein T can also be U.
 5. The polynucleotide of claim 1,wherein the polynucleotide is fully complementary to the nucleotideresidue number 805 through nucleotide residue number
 2493. 6. Thepolynucleotide of claim 1 comprising the sequence of a cDNA contained inthe plasmid designated p103P2D6-B deposited with American Type CultureCollection as Accession No. PTA-1895.
 7. The polynucleotide of claim 1comprising the sequence of a cDNA contained in the plasmid designatedp103P2D6-2 deposited with American Type Culture Collection as AccessionNo. PTA-1155.
 8. A polynucleotide that encodes the amino acid sequenceof SEQ ID NO:2.
 9. A recombinant expression vector comprising apolynucleotide of claim
 1. 10. An isolated host cell that contains anexpression vector of claim
 9. 11. The host cell of claim 10, wherein thehost cell comprises the plasmid designated p103P2D6-B deposited withAmerican Type Culture Collection as Accession No. PTA-1895.
 12. The hostcell of claim 10, wherein the host cell comprises the plasmid designatedp103P2D6-2 deposited with American Type Culture Collection as AccessionNo. PTA-1155.